Methods and compositions for producing fatty alcohols

ABSTRACT

Methods and compositions, including nucleotide sequences, amino acid sequences, and host cells, for producing fatty alcohols are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/109,131, filed Oct. 28, 2008, the entire contents of which are herebyincorporated by reference herein.

SEQUENCE LISTING

The replacement sequence listing submitted via EFS-Web, in compliancewith 37 C.F.R. 1.52(e)(2), is incorporated herein by reference. Thereplacement sequence listing text file submitted via EFS-Web containsthe file “SequenceListingLS16.txt”, created on May 17, 2010, which is974,190 bytes in size.

BACKGROUND OF THE INVENTION

Petroleum is a limited, natural resource found in the Earth in liquid,gaseous, or solid forms. Petroleum is primarily composed ofhydrocarbons, which are comprised mainly of carbon and hydrogen. It alsocontains significant amounts of other elements, such as, nitrogen,oxygen, or sulfur, in different forms.

Petroleum is a valuable resource, but petroleum products are developedat considerable costs, both financial and environmental. First, sourcesof petroleum must be discovered. Petroleum exploration is an expensiveand risky venture. The cost of exploring deep water wells can exceed$100 million. Moreover, there is no guarantee that these wells willcontain petroleum. It is estimated that only 40% of drilled wells leadto productive wells generating commercial hydrocarbons. In addition tothe economic cost, petroleum exploration carries a high environmentalcost. For example, offshore exploration disturbs the surrounding marineenvironments.

After a productive well is discovered, the petroleum must be extractedfrom the Earth at great expense. During primary recovery, the naturalpressure underground is sufficient to extract about 20% of the petroleumin the well. As this natural pressure falls, secondary recovery methodsare employed, if economical. Generally, secondary recovery involvesincreasing the well's pressure by, for example, water injection, naturalgas injection, or gas lift. Using secondary recovery methods, anadditional 5% to 15% of petroleum is recovered. Once secondary recoverymethods are exhausted, tertiary recovery methods can be used, ifeconomical. Tertiary methods involve reducing the viscosity of thepetroleum to make it easier to extract. Using tertiary recovery methods,an additional 5% to 15% of petroleum is recovered. Hence, even under thebest circumstances, only 50% of the petroleum in a well can beextracted. Petroleum extraction also carries an environmental cost. Forexample, petroleum extraction can result in large seepages of petroleumrising to the surface. Moreover, offshore drilling involves dredging theseabed which disrupts or destroys the surrounding marine environment.

Since petroleum deposits are not found uniformly throughout the Earth,petroleum must be transported over great distances from petroleumproducing regions to petroleum consuming regions. In addition to theshipping costs, there is also the environmental risk of devastating oilspills.

In its natural form, crude petroleum extracted from the Earth has fewcommercial uses. It is a mixture of hydrocarbons (e.g., paraffins (oralkanes), olefins (or alkenes), alkynes, napthenes (or cycloalkanes),aliphatic compounds, aromatic compounds, etc.) of varying length andcomplexity. In addition, crude petroleum contains other organiccompounds (e.g., organic compounds containing nitrogen, oxygen, sulfur,etc.) and impurities (e.g., sulfur, salt, acid, metals, etc.).

Hence, crude petroleum must be refined and purified before it can beused commercially. Due to its high energy density and its easytransportability, most petroleum is refined into fuels, such astransportation fuels (e.g., gasoline, diesel, aviation fuel, etc.),heating oil, liquefied petroleum gas, etc.

Crude petroleum is also a primary source of raw materials for producingpetrochemicals. The two main classes of raw materials derived frompetroleum are short chain olefins (e.g., ethylene and propylene) andaromatics (e.g., benzene and xylene isomers). These raw materials arederived from longer chain hydrocarbons in crude petroleum by cracking itat considerable expense using a variety of methods, such as catalyticcracking, steam cracking, or catalytic reforming. These raw materialsare used to make petrochemicals, which cannot be directly refined fromcrude petroleum, such as monomers, solvents, detergents, or adhesives.

One example of a raw material derived from crude petroleum is ethylene.Ethylene is used to produce petrochemicals, such as polyethylene,ethanol, ethylene oxide, ethylene glycol, polyester, glycol ether,ethoxylate, vinyl acetate, 1,2-dichloroethane, trichloroethylene,tetrachloroethylene, vinyl chloride, and polyvinyl chloride. Anadditional example of a raw material is propylene, which is used toproduce isopropyl alcohol, acrylonitrile, polypropylene, propyleneoxide, propylene glycol, glycol ethers, butylene, isobutylene,1,3-butadiene, synthetic elastomers, polyolefins, alpha-olefins, fattyalcohols, acrylic acid, acrylic polymers, allyl chloride,epichlorohydrin, and epoxy resins.

These petrochemicals can then be used to make specialty chemicals, suchas plastics, resins, fibers, elastomers, pharmaceuticals, lubricants, orgels. Particular specialty chemicals that can be produced frompetrochemical raw materials are fatty acids, hydrocarbons (e.g., longchain, branched chain, saturated, unsaturated, etc.), fatty alcohols,esters, fatty aldehydes, ketones, lubricants, etc.

Fatty alcohols have many commercial uses. Worldwide annual sales offatty alcohols and their derivatives are in excess of US$1 billion. Theshorter chain fatty alcohols are used in the cosmetic and foodindustries as emulsifiers, emollients, and thickeners. Due to theiramphiphilic nature, fatty alcohols behave as nonionic surfactants, whichare useful in personal care and household products, for example,detergents. In addition, fatty alcohols are used in waxes, gums, resins,pharmaceutical salves and lotions, lubricating oil additives, textileantistatic and finishing agents, plasticizers, cosmetics, industrialsolvents, and solvents for fats.

Aldehydes are used to produce many specialty chemicals. For example,aldehydes are used to produce polymers, resins (e.g., Bakelite), dyes,flavorings, plasticizers, perfumes, pharmaceuticals, and otherchemicals. Some are used as solvents, preservatives, or disinfectants.Some natural and synthetic compounds, such as vitamins and hormones, arealdehydes. In addition, many sugars contain aldehyde groups.

Obtaining these specialty chemicals from crude petroleum requires asignificant financial investment as well as a great deal of energy. Itis also an inefficient process because frequently the long chainhydrocarbons in crude petroleum are cracked to produce smaller monomers.These monomers are then used as the raw material to manufacture the morecomplex specialty chemicals.

In addition to the problems with exploring, extracting, transporting,and refining petroleum, petroleum is a limited and dwindling resource.One estimate of world petroleum consumption is 30 billion barrels peryear. By some estimates, it is predicted that at current productionlevels, the world's petroleum reserves could be depleted before the year2050.

Finally, the burning of petroleum based fuels releases greenhouse gases(e.g., carbon dioxide) and other forms of air pollution (e.g., carbonmonoxide, sulfur dioxide, etc.). As the world's demand for fuelincreases, the emission of greenhouse gases and other forms of airpollution also increases. The accumulation of greenhouse gases in theatmosphere can lead to an increase global warming. Hence, in addition todamaging the environment locally (e.g., oil spills, dredging of marineenvironments, etc.), burning petroleum also damages the environmentglobally.

Due to the inherent challenges posed by petroleum, there is a need for arenewable petroleum source that does not need to be explored, extracted,transported over long distances, or substantially refined likepetroleum. There is also a need for a renewable petroleum source whichcan be produced economically without creating the type of environmentaldamage produced by the petroleum industry and the burning of petroleumbased fuels. For similar reasons, there is also a need for a renewablesource of chemicals which are typically derived from petroleum.

One method of producing renewable petroleum is by engineeringmicroorganisms to produce renewable petroleum products. Somemicroorganisms have a natural ability to produce chemicals. For example,yeast has been used for centuries to produce ethanol (e.g., beer, wine,etc.). In recent years, through the development of advancedbiotechnologies, it is possible to metabolically engineer an organism toproduce bioproducts that were never previously produced. Products, suchas chemicals, derived from these cellular activities are known asbioproducts. Fuels produced these cellular activities are known asbiofuels. Biofuels are a renewable alternative fuel to petroleum basedfuels. Biofuels can be substituted for any petroleum based fuel (e.g.,gasoline, diesel, aviation fuel, heating oil, etc.). Biofuels can bederived from renewable sources, such as plant matter, animal matter, oreven waste products. These renewable sources are collectively known asbiomass. One advantage of biofuels over petroleum based fuels is thatthey do not require expensive and risky exploration or extraction. Inaddition, biofuels can be locally produced. Hence, they do not requiretransportation over long distances. Moreover, biofuels can be madedirectly without the need for expensive and energy intensive refining asis needed with refining crude petroleum. In other circumstances, thebiofuel may require a limited and cost-effective level of refining.Furthermore, the use of biofuels improves the environment by reducingthe amount of environmentally harmful emissions (e.g., green housegases, air pollution, etc.) released during combustion. For example,biofuels maintain a balanced carbon cycle because biofuels are producedfrom biomass, a renewable, natural resource. While the burning ofbiofuels will release carbon (e.g., as carbon dioxide), this carbon willbe recycled during the production of biomass (e.g., the cultivation ofcrops), thereby balancing the carbon cycle unlike petroleum based fuels.

For similar reasons, biologically derived chemicals offer the sameadvantages as biofuels over petroleum based fuels. Biologically derivedchemicals are a renewable alternative to petrochemicals. Biologicallyderived chemicals, such as hydrocarbons (e.g., alkanes, alkenes, oralkynes), fatty alcohols, esters, fatty acids, fatty aldehydes, andketones are superior to petrochemicals because they are produceddirectly without extensive refining. Unlike petrochemicals, biologicallyderived chemicals do not need to be refined like crude petroleum torecover raw materials which must then be further processed to make morecomplex petrochemicals. Biologically derived chemicals are directlyconverted from biomass to the desired chemical product.

SUMMARY OF THE INVENTION

The invention is based, at least in part, on the identification of genesthat encode fatty aldehyde biosynthetic polypeptides and fatty alcoholbiosynthetic polypeptides, which can be used to produce fatty aldehydesthat can subsequently be converted into fatty alcohols. Accordingly, inone aspect, the invention features a method of making a fatty alcohol.The method includes expressing in a host cell a gene encoding a fattyaldehyde biosynthetic polypeptide comprising the amino acid sequence ofSEQ ID NO:18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 264, 266, 268, 270, or 272, or a variant thereof. Insome embodiments, the method further includes isolating the fattyalcohol from the host cell. In some embodiments, the fatty alcohol ispresent in the extracellular environment. In certain embodiments, thefatty alcohol is isolated from the extracellular environment of the hostcell. In some embodiments, the fatty alcohol is secreted from the hostcell. In alternative embodiments, the fatty alcohol is transported intothe extracellular environment. In other embodiments, the fatty alcoholis passively transported into the extracellular environment.

In some embodiments, the fatty aldehyde biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 264, 266, 268,270, or 272 with one or more amino acid substitutions, additions,insertions, or deletions, and the polypeptide has carboxylic acidreductase activity. In some embodiments, the polypeptide has fatty acidreductase activity.

In some embodiments, the polypeptide comprises one or more of thefollowing conservative amino acid substitutions: replacement of analiphatic amino acid, such as alanine, valine, leucine, and isoleucine,with another aliphatic amino acid; replacement of a serine with athreonine; replacement of a threonine with a serine; replacement of anacidic residue, such as aspartic acid and glutamic acid, with anotheracidic residue; replacement of a residue bearing an amide group, such asasparagine and glutamine, with another residue bearing an amide group;exchange of a basic residue, such as lysine and arginine, with anotherbasic residue; and replacement of an aromatic residue, such asphenylalanine and tyrosine, with another aromatic residue. In someembodiments, the polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acidsubstitutions, additions, insertions, or deletions. In some embodiments,the polypeptide has carboxylic acid reductase activity. In someembodiments, the polypeptide has fatty acid reductase activity.

In some embodiments, the method further includes modifying theexpression of a gene encoding a fatty acid synthase in the host cell. Incertain embodiments, modifying the expression of a gene encoding a fattyacid synthase includes expressing a gene encoding a fatty acid synthasein the host cell and/or increasing the expression or activity of anendogenous fatty acid synthase in the host cell. In alternateembodiments, modifying the expression of a gene encoding a fatty acidsynthase includes attenuating a gene encoding a fatty acid synthase inthe host cell and/or decreasing the expression or activity of anendogenous fatty acid synthase in the host cell. In some embodiments,the fatty acid synthase is a thioesterase. In particular embodiments,the thioesterase is encoded by tesA, tesA without leader sequence, tesB,fatB, fatB2, fatB3, fatA, or fatA1.

In some embodiments, the method further includes expressing a geneencoding a fatty alcohol biosynthetic polypeptide in the host cell. Inparticular embodiments, the fatty alcohol biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, or 194, or a variant thereof.

In other embodiments, the host cell is genetically engineered to expressan attenuated level of a fatty acid degradation enzyme relative to awild type host cell. In some embodiments, the host cell is geneticallyengineered to express an attenuated level of an acyl-CoA synthaserelative to a wild type host cell. In particular embodiments, the hostcell expresses an attenuated level of an acyl-CoA synthase encoded byfadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.In certain embodiments, the genetically engineered host cell comprises aknockout of one or more genes encoding a fatty acid degradation enzyme,such as the aforementioned acyl-CoA synthase genes.

In yet other embodiments, the host cell is genetically engineered toexpress an attenuated level of a dehydratase/isomerase enzyme, such asan enzyme encoded by fabA or by a gene listed in FIG. 15. In someembodiments, the host cell comprises a knockout of fabA or a gene listedin FIG. 15. In other embodiments, the host cell is geneticallyengineered to express an attenuated level of a ketoacyl-ACP synthase,such as an enzyme encoded by fabB or by a gene listed in FIG. 16. Incertain embodiments, the host cell comprises a knockout of fabB or agene listed in FIG. 16. In yet other embodiments, the host cell isgenetically engineered to express a modified level of a gene encoding adesaturase enzyme, such as desA.

In some embodiments, the polypeptide is from a bacterium, a plant, aninsect, a yeast, a fungus, or a mammal.

In certain embodiments, the polypeptide is from a mammalian cell, plantcell, insect cell, yeast cell, fungus cell, filamentous fungi cell,bacterial cell, or any other organism described herein. In someembodiments, the bacterium is a mycobacterium selected from the groupconsisting of Mycobacterium smegmatis, Mycobacterium abscessus,Mycobacterium avium, Mycobacterium bovis, Mycobacterium tuberculosis,Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium ulcerans.In other embodiments, the bacterium is Nocardia sp. NRRL 5646, Nocardiafarcinica, Streptomyces griseus, Salinispora arenicola, or Clavibactermichiganenesis.

In some embodiments, the method further includes culturing the host cellin the presence of at least one biological substrate for the fattyaldehyde biosynthetic polypeptide.

In another aspect, the invention features a method of producing a fattyalcohol. The method includes expressing in a host cell a gene encoding afatty aldehyde biosynthetic polypeptide comprising an amino acidsequence having at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% sequence identity to the amino acid sequence of SEQ IDNO:18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 264, 266, 268, 270, or 272. In some embodiments, the aminoacid sequence is the amino acid sequence of SEQ ID NO:18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 264,266, 268, 270, or 272.

In some embodiments, the method further includes isolating the fattyalcohol from the host cell. In some embodiments, the fatty alcohol ispresent in the extracellular environment. In certain embodiments, thefatty alcohol is isolated from the extracellular environment of the hostcell. In some embodiments, the fatty alcohol is secreted from the hostcell. In alternative embodiments, the fatty alcohol is transported intothe extracellular environment. In other embodiments, the fatty alcoholis passively transported into the extracellular environment.

In some embodiments, the method further includes modifying theexpression of a gene encoding a fatty acid synthase in the host cell. Incertain embodiments, modifying the expression of a gene encoding a fattyacid synthase includes expressing a gene encoding a fatty acid synthasein the host cell and/or increasing the expression or activity of anendogenous fatty acid synthase in the host cell. In alternateembodiments, modifying the expression of a gene encoding a fatty acidsynthase includes attenuating a gene encoding a fatty acid synthase inthe host cell and/or decreasing the expression or activity of anendogenous fatty acid synthase in the host cell. In some embodiments,the fatty acid synthase is a thioesterase. In particular embodiments,the thioesterase is encoded by tesA, tesA without leader sequence, tesB,fatB, fatB2, fatB3, fatA, or fatA1.

In some embodiments, the method further includes expressing a geneencoding a fatty alcohol biosynthetic polypeptide in the host cell. Inparticular embodiments, the fatty alcohol biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, or 194, or a variant thereof.

In other embodiments, the host cell is genetically engineered to expressan attenuated level of a fatty acid degradation enzyme relative to awild type host cell. In some embodiments, the host cell is geneticallyengineered to express an attenuated level of an acyl-CoA synthaserelative to a wild type host cell. In particular embodiments, the hostcell expresses an attenuated level of an acyl-CoA synthase encoded byfadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.In certain embodiments, the genetically engineered host cell comprises aknockout of one or more genes encoding a fatty acid degradation enzyme,such as the aforementioned acyl-CoA synthase genes.

In yet other embodiments, the host cell is genetically engineered toexpress an attenuated level of a dehydratase/isomerase enzyme, such asan enzyme encoded by fabA or by a gene listed in FIG. 15. In someembodiments, the host cell comprises a knockout of fabA or a gene listedin FIG. 15. In other embodiments, the host cell is geneticallyengineered to express an attenuated level of a ketoacyl-ACP synthase,such as an enzyme encoded by fabB or by a gene listed in FIG. 16. Incertain embodiments, the host cell comprises a knockout of fabB or agene listed in FIG. 16. In yet other embodiments, the host cell isgenetically engineered to express a modified level of a gene encoding adesaturase enzyme, such as desA.

In some embodiments, the polypeptide is from a bacterium, a plant, aninsect, a yeast, a fungus, or a mammal.

In certain embodiments, the polypeptide is from a mammalian cell, plantcell, insect cell, yeast cell, fungus cell, filamentous fungi cell,bacterial cell, or any other organism described herein. In someembodiments, the bacterium is a mycobacterium selected from the groupconsisting of Mycobacterium smegmatis, Mycobacterium abscessus,Mycobacterium avium, Mycobacterium bovis, Mycobacterium tuberculosis,Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium ulcerans.In other embodiments, the bacterium is Nocardia sp. NRRL 5646, Nocardiafarcinica, Streptomyces griseus, Salinispora arenicola, or Clavibactermichiganenesis.

In some embodiments, the method further includes culturing the host cellin the presence of at least one biological substrate for the fattyaldehyde biosynthetic polypeptide.

In another aspect, the invention features a method of producing a fattyalcohol. The method includes expressing in a host cell a polynucleotidethat hybridizes to a complement of the nucleotide sequence of SEQ IDNO:17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 263, 265, 267, 269, or 271, or to a fragment thereof,wherein the polynucleotide encodes a polypeptide having carboxylic acidreductase activity. In some embodiments, the polypeptide has fatty acidreductase activity.

In some embodiments, the method further includes isolating the fattyalcohol from the host cell. In some embodiments, the fatty alcohol ispresent in the extracellular environment. In certain embodiments, thefatty alcohol is isolated from the extracellular environment of the hostcell. In some embodiments, the fatty alcohol is secreted from the hostcell. In alternative embodiments, the fatty alcohol is transported intothe extracellular environment. In other embodiments, the fatty alcoholis passively transported into the extracellular environment.

In some embodiments, the polynucleotide hybridizes under low stringency,medium stringency, high stringency, or very high stringency conditions,to a complement of the nucleotide sequence of SEQ ID NO:17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 263,265, 267, 269, or 271, or to a fragment thereof.

In some embodiments, the method further includes modifying theexpression of a gene encoding a fatty acid synthase in the host cell. Incertain embodiments, modifying the expression of a gene encoding a fattyacid synthase includes expressing a gene encoding a fatty acid synthasein the host cell and/or increasing the expression or activity of anendogenous fatty acid synthase in the host cell. In alternateembodiments, modifying the expression of a gene encoding a fatty acidsynthase includes attenuating a gene encoding a fatty acid synthase inthe host cell and/or decreasing the expression or activity of anendogenous fatty acid synthase in the host cell. In some embodiments,the fatty acid synthase is a thioesterase. In particular embodiments,the thioesterase is encoded by tesA, tesA without leader sequence, tesB,fatB, fatB2, fatB3, fatA, or fatA1.

In some embodiments, the method further includes expressing a geneencoding a fatty alcohol biosynthetic polypeptide in the host cell. Inparticular embodiments, the fatty alcohol biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, or 194, or a variant thereof.

In other embodiments, the host cell is genetically engineered to expressan attenuated level of a fatty acid degradation enzyme relative to awild type host cell. In some embodiments, the host cell is geneticallyengineered to express an attenuated level of an acyl-CoA synthaserelative to a wild type host cell. In particular embodiments, the hostcell expresses an attenuated level of an acyl-CoA synthase encoded byfadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.In certain embodiments, the genetically engineered host cell comprises aknockout of one or more genes encoding a fatty acid degradation enzyme,such as the aforementioned acyl-CoA synthase genes.

In yet other embodiments, the host cell is genetically engineered toexpress an attenuated level of a dehydratase/isomerase enzyme, such asan enzyme encoded by fabA or by a gene listed in FIG. 15. In someembodiments, the host cell comprises a knockout of fabA or a gene listedin FIG. 15. In other embodiments, the host cell is geneticallyengineered to express an attenuated level of a ketoacyl-ACP synthase,such as an enzyme encoded by fabB or by a gene listed in FIG. 16. Incertain embodiments, the host cell comprises a knockout of fabB or agene listed in FIG. 16. In yet other embodiments, the host cell isgenetically engineered to express a modified level of a gene encoding adesaturase enzyme, such as desA.

In some embodiments, the polynucleotide is from a bacterium, a plant, aninsect, a yeast, a fungus, or a mammal.

In certain embodiments, the polypeptide is from a mammalian cell, plantcell, insect cell, yeast cell, fungus cell, filamentous fungi cell,bacterial cell, or any other organism described herein. In someembodiments, the bacterium is a mycobacterium selected from the groupconsisting of Mycobacterium smegmatis, Mycobacterium abscessus,Mycobacterium avium, Mycobacterium bovis, Mycobacterium tuberculosis,Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium ulcerans.In other embodiments, the bacterium is Nocardia sp. NRRL 5646, Nocardiafarcinica, Streptomyces griseus, Salinispora arenicola, or Clavibactermichiganenesis.

In some embodiments, the method further includes culturing the host cellin the presence of at least one biological substrate for the fattyaldehyde biosynthetic polypeptide.

In another aspect, the invention features a method of producing a fattyalcohol. The method comprises expressing in a host cell a gene encodinga fatty aldehyde biosynthetic polypeptide comprising the amino acid ofSEQ ID NO:16, or a variant thereof. In some embodiments, the methodfurther includes isolating the fatty alcohol from the host cell. In someembodiments, the fatty alcohol is present in the extracellularenvironment. In certain embodiments, the fatty alcohol is isolated fromthe extracellular environment of the host cell. In some embodiments, thefatty alcohol is secreted from the host cell. In alternativeembodiments, the fatty alcohol is transported into the extracellularenvironment. In other embodiments, the fatty alcohol is passivelytransported into the extracellular environment.

In some embodiments, the polypeptide comprises the amino acid sequenceof SEQ ID NO:16 with one or more amino acid substitutions, additions,insertions, or deletions, wherein the polypeptide has carboxylic acidreductase activity. In some embodiments, the polypeptide has fatty acidreductase activity.

In some embodiments, the polypeptide comprises one or more of thefollowing conservative amino acid substitutions: replacement of analiphatic amino acid, such as alanine, valine, leucine, and isoleucine,with another aliphatic amino acid; replacement of a serine with athreonine; replacement of a threonine with a serine; replacement of anacidic residue, such as aspartic acid and glutamic acid, with anotheracidic residue; replacement of a residue bearing an amide group, such asasparagine and glutamine, with another residue bearing an amide group;exchange of a basic residue, such as lysine and arginine, with anotherbasic residue; and replacement of an aromatic residue, such asphenylalanine and tyrosine, with another aromatic residue. In someembodiments, the polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acidsubstitutions, additions, insertions, or deletions. In some embodiments,the polypeptide has carboxylic acid reductase activity. In someembodiments, the polypeptide has fatty acid reductase activity.

In some embodiments, the method further includes modifying theexpression of a gene encoding a fatty acid synthase in the host cell. Incertain embodiments, modifying the expression of a gene encoding a fattyacid synthase includes expressing a gene encoding a fatty acid synthasein the host cell and/or increasing the expression or activity of anendogenous fatty acid synthase in the host cell. In alternateembodiments, modifying the expression of a gene encoding a fatty acidsynthase includes attenuating a gene encoding a fatty acid synthase inthe host cell and/or decreasing the expression or activity of anendogenous fatty acid synthase in the host cell. In some embodiments,the fatty acid synthase is a thioesterase. In particular embodiments,the thioesterase is encoded by tesA, tesA without leader sequence, tesB,fatB, fatB2, fatB3, fatA, or fatA1.

In some embodiments, the method further includes expressing a geneencoding a fatty alcohol biosynthetic polypeptide in the host cell. Inparticular embodiments, the fatty alcohol biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, or 194, or a variant thereof.

In other embodiments, the host cell is genetically engineered to expressan attenuated level of a fatty acid degradation enzyme relative to awild type host cell. In some embodiments, the host cell is geneticallyengineered to express an attenuated level of an acyl-CoA synthaserelative to a wild type host cell. In particular embodiments, the hostcell expresses an attenuated level of an acyl-CoA synthase encoded byfadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.In certain embodiments, the genetically engineered host cell comprises aknockout of one or more genes encoding a fatty acid degradation enzyme,such as the aforementioned acyl-CoA synthase genes.

In yet other embodiments, the host cell is genetically engineered toexpress an attenuated level of a dehydratase/isomerase enzyme, such asan enzyme encoded by fabA or by a gene listed in FIG. 15. In someembodiments, the host cell comprises a knockout of fabA or a gene listedin FIG. 15. In other embodiments, the host cell is geneticallyengineered to express an attenuated level of a ketoacyl-ACP synthase,such as an enzyme encoded by fabB or by a gene listed in FIG. 16. Incertain embodiments, the host cell comprises a knockout of fabB or agene listed in FIG. 16. In yet other embodiments, the host cell isgenetically engineered to express a modified level of a gene encoding adesaturase enzyme, such as desA.

In some embodiments, the method further includes culturing the host cellin the presence of at least one biological substrate for the fattyaldehyde biosynthetic polypeptide.

In another aspect, the invention features a method of producing a fattyalcohol. The method includes expressing in a host cell a gene encoding afatty aldehyde biosynthetic polypeptide comprising an amino acidsequence having at least about 70% sequence identity to the amino acidsequence of SEQ ID NO:16.

In some embodiments, the method further includes isolating the fattyalcohol from the host cell. In certain embodiments, the fatty alcohol isisolated from the extracellular environment of the host cell. In someembodiments, the fatty alcohol is secreted from the host cell.

In some embodiments, the amino acid sequence has at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% sequence identity to the amino acid sequenceof SEQ ID NO:16. In some embodiments, the amino acid sequence is SEQ IDNO:16.

In some embodiments, the method further includes modifying theexpression of a gene encoding a fatty acid synthase in the host cell. Incertain embodiments, modifying the expression of a gene encoding a fattyacid synthase includes expressing a gene encoding a fatty acid synthasein the host cell and/or increasing the expression or activity of anendogenous fatty acid synthase in the host cell. In alternateembodiments, modifying the expression of a gene encoding a fatty acidsynthase includes attenuating a gene encoding a fatty acid synthase inthe host cell and/or decreasing the expression or activity of anendogenous fatty acid synthase in the host cell. In some embodiments,the fatty acid synthase is a thioesterase. In particular embodiments,the thioesterase is encoded by tesA, tesA without leader sequence, tesB,fatB, fatB2, fatB3, fatA, or fatA1.

In some embodiments, the method further includes expressing a geneencoding a fatty alcohol biosynthetic polypeptide in the host cell. Inparticular embodiments, the fatty alcohol biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, or 194, or a variant thereof.

In other embodiments, the host cell is genetically engineered to expressan attenuated level of a fatty acid degradation enzyme relative to awild type host cell. In some embodiments, the host cell is geneticallyengineered to express an attenuated level of an acyl-CoA synthaserelative to a wild type host cell. In particular embodiments, the hostcell expresses an attenuated level of an acyl-CoA synthase encoded byfadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.In certain embodiments, the genetically engineered host cell comprises aknockout of one or more genes encoding a fatty acid degradation enzyme,such as the aforementioned acyl-CoA synthase genes.

In yet other embodiments, the host cell is genetically engineered toexpress an attenuated level of a dehydratase/isomerase enzyme, such asan enzyme encoded by fabA or by a gene listed in FIG. 15. In someembodiments, the host cell comprises a knockout of fabA or a gene listedin FIG. 15. In other embodiments, the host cell is geneticallyengineered to express an attenuated level of a ketoacyl-ACP synthase,such as an enzyme encoded by fabB or by a gene listed in FIG. 16. Incertain embodiments, the host cell comprises a knockout of fabB or agene listed in FIG. 16. In yet other embodiments, the host cell isgenetically engineered to express a modified level of a gene encoding adesaturase enzyme, such as desA.

In some embodiments, the method further includes culturing the host cellin the presence of at least one biological substrate for the fattyaldehyde biosynthetic polypeptide.

In another aspect, the invention features a method of producing a fattyalcohol. The method includes expressing in a host cell a polynucleotidethat hybridizes to a complement of the nucleotide sequence of SEQ IDNO:15, or to a fragment thereof, wherein the polynucleotide encodes apolypeptide having carboxylic acid reductase activity. In someembodiments, the polypeptide has fatty acid reductase activity.

In some embodiments, the method further includes modifying theexpression of a gene encoding a fatty acid synthase in the host cell. Incertain embodiments, modifying the expression of a gene encoding a fattyacid synthase includes expressing a gene encoding a fatty acid synthasein the host cell and/or increasing the expression or activity of anendogenous fatty acid synthase in the host cell. In alternateembodiments, modifying the expression of a gene encoding a fatty acidsynthase includes attenuating a gene encoding a fatty acid synthase inthe host cell and/or decreasing the expression or activity of anendogenous fatty acid synthase in the host cell. In some embodiments,the fatty acid synthase is a thioesterase. In particular embodiments,the thioesterase is encoded by tesA, tesA without leader sequence, tesB,fatB, fatB2, fatB3, fatA, or fatA1.

In some embodiments, the method further includes expressing a geneencoding a fatty alcohol biosynthetic polypeptide in the host cell. Inparticular embodiments, the fatty alcohol biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, or 194, or a variant thereof.

In other embodiments, the host cell is genetically engineered to expressan attenuated level of a fatty acid degradation enzyme relative to awild type host cell. In some embodiments, the host cell is geneticallyengineered to express an attenuated level of an acyl-CoA synthaserelative to a wild type host cell. In particular embodiments, the hostcell expresses an attenuated level of an acyl-CoA synthase encoded byfadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.In certain embodiments, the genetically engineered host cell comprises aknockout of one or more genes encoding a fatty acid degradation enzyme,such as the aforementioned acyl-CoA synthase genes.

In yet other embodiments, the host cell is genetically engineered toexpress an attenuated level of a dehydratase/isomerase enzyme, such asan enzyme encoded by fabA or by a gene listed in FIG. 15. In someembodiments, the host cell comprises a knockout of fabA or a gene listedin FIG. 15. In other embodiments, the host cell is geneticallyengineered to express an attenuated level of a ketoacyl-ACP synthase,such as an enzyme encoded by fabB or by a gene listed in FIG. 16. Incertain embodiments, the host cell comprises a knockout of fabB or agene listed in FIG. 16. In yet other embodiments, the host cell isgenetically engineered to express a modified level of a gene encoding adesaturase enzyme, such as desA.

In some embodiments, the method further includes isolating the fattyalcohol from the host cell. In some embodiments, the fatty alcohol ispresent in the extracellular environment. In certain embodiments, thefatty alcohol is isolated from the extracellular environment of the hostcell. In some embodiments, the fatty alcohol is secreted from the hostcell. In alternative embodiments, the fatty alcohol is transported intothe extracellular environment. In other embodiments, the fatty alcoholis passively transported into the extracellular environment.

In some embodiments, the polynucleotide hybridizes under low stringency,medium stringency, high stringency, or very high stringency conditions,to a complement of the nucleotide sequence of SEQ ID NO:15, or to afragment thereof.

In some embodiments, the method further includes culturing the host cellin the presence of at least one biological substrate for the fattyaldehyde biosynthetic polypeptide.

In another aspect, the invention features a method of producing a fattyalcohol. The method includes expressing in a host cell a recombinantvector comprising a fatty aldehyde biosynthetic nucleotide sequencehaving at least about 70% sequence identity to a nucleotide sequencelisted in FIG. 8. In some embodiments, the nucleotide sequence has atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity to thenucleotide sequence of SEQ ID NO:17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 263, 265, 267, 269, or 271. Insome embodiments, the nucleotide sequence is SEQ ID NO:17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 263,265, 267, 269, or 271.

In some embodiments, the method further includes isolating the fattyalcohol from the host cell. In some embodiments, the fatty alcohol ispresent in the extracellular environment. In certain embodiments, thefatty alcohol is isolated from the extracellular environment of the hostcell. In some embodiments, the fatty alcohol is secreted from the hostcell. In alternative embodiments, the fatty alcohol is transported intothe extracellular environment. In other embodiments, the fatty alcoholis passively transported into the extracellular environment.

In some embodiments, the recombinant vector further comprises a promoteroperably linked to the nucleotide sequence. In certain embodiments, thepromoter is a developmentally-regulated, an organelle-specific, atissue-specific, an inducible, a constitutive, or a cell-specificpromoter.

In other embodiments, the recombinant vector comprises at least onesequence selected from the group consisting of (a) a regulatory sequenceoperatively coupled to the nucleotide sequence; (b) a selection markeroperatively coupled to the nucleotide sequence; (c) a marker sequenceoperatively coupled to the nucleotide sequence; (d) a purificationmoiety operatively coupled to the nucleotide sequence; (e) a secretionsequence operatively coupled to the nucleotide sequence; and (f) atargeting sequence operatively coupled to the nucleotide sequence.

In some embodiments, the recombinant vector is a plasmid.

In some embodiments, the host cell expresses a polypeptide encoded bythe recombinant vector. In some embodiments, the nucleotide sequence isstably incorporated into the genomic DNA of the host cell, and theexpression of the nucleotide sequence is under the control of aregulated promoter region.

In some embodiments, the method further includes modifying theexpression of a gene encoding a fatty acid synthase in the host cell. Incertain embodiments, modifying the expression of a gene encoding a fattyacid synthase includes expressing a gene encoding a fatty acid synthasein the host cell and/or increasing the expression or activity of anendogenous fatty acid synthase in the host cell. In alternateembodiments, modifying the expression of a gene encoding a fatty acidsynthase includes attenuating a gene encoding a fatty acid synthase inthe host cell and/or decreasing the expression or activity of anendogenous fatty acid synthase in the host cell. In some embodiments,the fatty acid synthase is a thioesterase. In particular embodiments,the thioesterase is encoded by tesA, tesA without leader sequence, tesB,fatB, fatB2, fatB3, fatA, or fatA1.

In some embodiments, the method further includes expressing a geneencoding a fatty alcohol biosynthetic polypeptide in the host cell. Inparticular embodiments, the fatty alcohol biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, or 194, or a variant thereof.

In other embodiments, the host cell is genetically engineered to expressan attenuated level of a fatty acid degradation enzyme relative to awild type host cell. In some embodiments, the host cell is geneticallyengineered to express an attenuated level of an acyl-CoA synthaserelative to a wild type host cell. In particular embodiments, the hostcell expresses an attenuated level of an acyl-CoA synthase encoded byfadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.In certain embodiments, the genetically engineered host cell comprises aknockout of one or more genes encoding a fatty acid degradation enzyme,such as the aforementioned acyl-CoA synthase genes.

In yet other embodiments, the host cell is genetically engineered toexpress an attenuated level of a dehydratase/isomerase enzyme, such asan enzyme encoded by fabA or by a gene listed in FIG. 15. In someembodiments, the host cell comprises a knockout of fabA or a gene listedin FIG. 15. In other embodiments, the host cell is geneticallyengineered to express an attenuated level of a ketoacyl-ACP synthase,such as an enzyme encoded by fabB or by a gene listed in FIG. 16. Incertain embodiments, the host cell comprises a knockout of fabB or agene listed in FIG. 16. In yet other embodiments, the host cell isgenetically engineered to express a modified level of a gene encoding adesaturase enzyme, such as desA.

In some embodiments, the method further includes culturing the host cellin the presence of at least one biological substrate for a fattyaldehyde biosynthetic polypeptide.

In another aspect, the invention features a method of producing a fattyalcohol. The method includes expressing in a host cell a recombinantvector comprising a fatty aldehyde biosynthetic nucleotide sequencehaving at least about 70% sequence identity to the nucleotide sequenceof SEQ ID NO:15.

In some embodiments, the method further includes isolating the fattyalcohol from the host cell. In some embodiments, the fatty alcohol ispresent in the extracellular environment. In certain embodiments, thefatty alcohol is isolated from the extracellular environment of the hostcell. In some embodiments, the fatty alcohol is secreted from the hostcell. In alternative embodiments, the fatty alcohol is transported intothe extracellular environment. In other embodiments, the fatty alcoholis passively transported into the extracellular environment.

In some embodiments, the nucleotide sequence has at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% sequence identity to the nucleotide sequenceof SEQ ID NO:15. In some embodiments, the nucleotide sequence is thenucleotide sequence of SEQ ID NO:15.

In some embodiments, the method further includes modifying theexpression of a gene encoding a fatty acid synthase in the host cell. Incertain embodiments, modifying the expression of a gene encoding a fattyacid synthase includes expressing a gene encoding a fatty acid synthasein the host cell and/or increasing the expression or activity of anendogenous fatty acid synthase in the host cell. In alternateembodiments, modifying the expression of a gene encoding a fatty acidsynthase includes attenuating a gene encoding a fatty acid synthase inthe host cell and/or decreasing the expression or activity of anendogenous fatty acid synthase in the host cell. In some embodiments,the fatty acid synthase is a thioesterase. In particular embodiments,the thioesterase is encoded by tesA, tesA without leader sequence, tesB,fatB, fatB2, fatB3, fatA, or fatA1.

In some embodiments, the method further includes expressing a geneencoding a fatty alcohol biosynthetic polypeptide in the host cell. Inparticular embodiments, the fatty alcohol biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, or 194, or a variant thereof.

In other embodiments, the host cell is genetically engineered to expressan attenuated level of a fatty acid degradation enzyme relative to awild type host cell. In some embodiments, the host cell is geneticallyengineered to express an attenuated level of an acyl-CoA synthaserelative to a wild type host cell. In particular embodiments, the hostcell expresses an attenuated level of an acyl-CoA synthase encoded byfadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.In certain embodiments, the genetically engineered host cell comprises aknockout of one or more genes encoding a fatty acid degradation enzyme,such as the aforementioned acyl-CoA synthase genes.

In yet other embodiments, the host cell is genetically engineered toexpress an attenuated level of a dehydratase/isomerase enzyme, such asan enzyme encoded by fabA or by a gene listed in FIG. 15. In someembodiments, the host cell comprises a knockout of fabA or a gene listedin FIG. 15. In other embodiments, the host cell is geneticallyengineered to express an attenuated level of a ketoacyl-ACP synthase,such as an enzyme encoded by fabB or by a gene listed in FIG. 16. Incertain embodiments, the host cell comprises a knockout of fabB or agene listed in FIG. 16. In yet other embodiments, the host cell isgenetically engineered to express a modified level of a gene encoding adesaturase enzyme, such as desA.

In some embodiments, the recombinant vector further comprises a promoteroperably linked to the nucleotide sequence. In certain embodiments, thepromoter is a developmentally-regulated, an organelle-specific, atissue-specific, an inducible, a constitutive, or a cell-specificpromoter.

In other embodiments, the recombinant vector comprises at least onesequence selected from the group consisting of (a) a regulatory sequenceoperatively coupled to the nucleotide sequence; (b) a selection markeroperatively coupled to the nucleotide sequence; (c) a marker sequenceoperatively coupled to the nucleotide sequence; (d) a purificationmoiety operatively coupled to the nucleotide sequence; (e) a secretionsequence operatively coupled to the nucleotide sequence; and (f) atargeting sequence operatively coupled to the nucleotide sequence.

In some embodiments, the recombinant vector is a plasmid.

In some embodiments, the host cell expresses a polypeptide encoded bythe recombinant vector. In some embodiments, the nucleotide sequence isstably incorporated into the genomic DNA of the host cell, and theexpression of the nucleotide sequence is under the control of aregulated promoter region.

In some embodiments, the method further includes culturing the host cellin the presence of at least one biological substrate for a fattyaldehyde biosynthetic polypeptide.

In another aspect, the invention features a method of producing a fattyalcohol. The method includes expressing in a host cell a gene encoding afatty aldehyde biosynthetic polypeptide comprising (i) SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, and SEQ ID NO:10; (ii) SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, or SEQ ID NO:14; and/or (iii) SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:10, and SEQ ID NO:11; wherein the polypeptide has carboxylicacid reductase activity. In some embodiments, the polypeptide has fattyacid reductase activity.

In some embodiments, the method further includes isolating the fattyalcohol from the host cell. In some embodiments, the fatty alcohol ispresent in the extracellular environment. In certain embodiments, thefatty alcohol is isolated from the extracellular environment of the hostcell. In some embodiments, the fatty alcohol is secreted from the hostcell. In alternative embodiments, the fatty alcohol is transported intothe extracellular environment. In other embodiments, the fatty alcoholis passively transported into the extracellular environment.

In some embodiments, the polypeptide is about 1,000 amino acids to about2,000 amino acids in length. In certain embodiments, the polypeptide isabout 1,000 amino acids in length, about 1,050 amino acids in length,about 1,100 amino acids in length, about 1,150 amino acids in length,about 1,200 amino acids in length, about 1,250 amino acids in length,about 1,300 amino acids in length, about 1,400 amino acids in length,about 1,500 amino acids in length, about 1,600 amino acids in length,about 1,700 amino acids in length, about 1,800 amino acids in length,about 1,900 amino acids in length, or about 2,000 amino acids in length.In other embodiments, the polypeptide is up to about 2,000 amino acidsin length, up to about 1,900 amino acids in length, up to about 1,800amino acids in length, up to about 1,700 amino acids in length, up toabout 1,600 amino acids in length, up to about 1,500 amino acids inlength, up to about 1,400 amino acids in length, up to about 1,300 aminoacids in length, up to about 1,250 amino acids in length, up to about1,200 amino acids in length, up to about 1,150 amino acids in length, upto about 1,100 amino acids in length, up to about 1,050 amino acids inlength, or up to about 1,000 amino acids in length. In otherembodiments, the polypeptide is more than about 1,000 amino acids inlength, more than about 1,050 amino acids in length, more than about1,100 amino acids in length, more than about 1,150 amino acids inlength, more than about 1,200 amino acids in length, more than about1,250 amino acids in length, more than about 1,300 amino acids inlength, more than about 1,400 amino acids in length, more than about1,500 amino acids in length, more than about 1,600 amino acids inlength, more than about 1,700 amino acids in length, more than about1,800 amino acids in length, more than about 1,900 amino acids inlength, or about 2,000 amino acids in length.

In some embodiments, the method further includes modifying theexpression of a gene encoding a fatty acid synthase in the host cell. Incertain embodiments, modifying the expression of a gene encoding a fattyacid synthase includes expressing a gene encoding a fatty acid synthasein the host cell and/or increasing the expression or activity of anendogenous fatty acid synthase in the host cell. In alternateembodiments, modifying the expression of a gene encoding a fatty acidsynthase includes attenuating a gene encoding a fatty acid synthase inthe host cell and/or decreasing the expression or activity of anendogenous fatty acid synthase in the host cell. In some embodiments,the fatty acid synthase is a thioesterase. In particular embodiments,the thioesterase is encoded by tesA, tesA without leader sequence, tesB,fatB, fatB2, fatB3, fatA, or fatA1.

In some embodiments, the method further includes expressing a geneencoding a fatty alcohol biosynthetic polypeptide in the host cell. Inparticular embodiments, the fatty alcohol biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, or 194, or a variant thereof.

In other embodiments, the host cell is genetically engineered to expressan attenuated level of a fatty acid degradation enzyme relative to awild type host cell. In some embodiments, the host cell is geneticallyengineered to express an attenuated level of an acyl-CoA synthaserelative to a wild type host cell. In particular embodiments, the hostcell expresses an attenuated level of an acyl-CoA synthase encoded byfadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.In certain embodiments, the genetically engineered host cell comprises aknockout of one or more genes encoding a fatty acid degradation enzyme,such as the aforementioned acyl-CoA synthase genes.

In yet other embodiments, the host cell is genetically engineered toexpress an attenuated level of a dehydratase/isomerase enzyme, such asan enzyme encoded by fabA or by a gene listed in FIG. 15. In someembodiments, the host cell comprises a knockout of fabA or a gene listedin FIG. 15. In other embodiments, the host cell is geneticallyengineered to express an attenuated level of a ketoacyl-ACP synthase,such as an enzyme encoded by fabB or by a gene listed in FIG. 16. Incertain embodiments, the host cell comprises a knockout of fabB or agene listed in FIG. 16. In yet other embodiments, the host cell isgenetically engineered to express a modified level of a gene encoding adesaturase enzyme, such as desA.

In some embodiments, the method further includes culturing the host cellin the presence of at least one biological substrate for the fattyaldehyde biosynthetic polypeptide.

In another aspect, the invention features a method of making a fattyalcohol. The method includes expressing in a host cell a gene encoding afatty alcohol biosynthetic polypeptide comprising the amino acidsequence of SEQ ID NO:94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, or 194, or avariant thereof. In some embodiments, the method further includesisolating the fatty alcohol from the host cell. In some embodiments, thefatty alcohol is present in the extracellular environment. In certainembodiments, the fatty alcohol is isolated from the extracellularenvironment of the host cell. In some embodiments, the fatty alcohol issecreted from the host cell. In alternative embodiments, the fattyalcohol is transported into the extracellular environment. In otherembodiments, the fatty alcohol is passively transported into theextracellular environment.

In some embodiments, the fatty alcohol biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, or 194 with one or more amino acid substitutions,additions, insertions, or deletions, and the polypeptide has alcoholdehydrogenase activity.

In some embodiments, the polypeptide comprises one or more of thefollowing conservative amino acid substitutions: replacement of analiphatic amino acid, such as alanine, valine, leucine, and isoleucine,with another aliphatic amino acid; replacement of a serine with athreonine; replacement of a threonine with a serine; replacement of anacidic residue, such as aspartic acid and glutamic acid, with anotheracidic residue; replacement of a residue bearing an amide group, such asasparagine and glutamine, with another residue bearing an amide group;exchange of a basic residue, such as lysine and arginine, with anotherbasic residue; and replacement of an aromatic residue, such asphenylalanine and tyrosine, with another aromatic residue. In someembodiments, the polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acidsubstitutions, additions, insertions, or deletions. In some embodiments,the polypeptide has alcohol dehydrogenase activity.

In some embodiments, the method further includes modifying theexpression of a gene encoding a fatty acid synthase in the host cell. Incertain embodiments, modifying the expression of a gene encoding a fattyacid synthase includes expressing a gene encoding a fatty acid synthasein the host cell and/or increasing the expression or activity of anendogenous fatty acid synthase in the host cell. In alternateembodiments, modifying the expression of a gene encoding a fatty acidsynthase includes attenuating a gene encoding a fatty acid synthase inthe host cell and/or decreasing the expression or activity of anendogenous fatty acid synthase in the host cell. In some embodiments,the fatty acid synthase is a thioesterase. In particular embodiments,the thioesterase is encoded by tesA, tesA without leader sequence, tesB,fatB, fatB2, fatB3, fatA, or fatA1.

In some embodiments, the method further includes expressing a geneencoding a fatty aldehyde biosynthetic polypeptide in the host cell. Inparticular embodiments, the fatty aldehyde biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 264, 266, 268,270, or 272, or a variant thereof.

In other embodiments, the host cell is genetically engineered to expressan attenuated level of a fatty acid degradation enzyme relative to awild type host cell. In some embodiments, the host cell is geneticallyengineered to express an attenuated level of an acyl-CoA synthaserelative to a wild type host cell. In particular embodiments, the hostcell expresses an attenuated level of an acyl-CoA synthase encoded byfadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.In certain embodiments, the genetically engineered host cell comprises aknockout of one or more genes encoding a fatty acid degradation enzyme,such as the aforementioned acyl-CoA synthase genes.

In yet other embodiments, the host cell is genetically engineered toexpress an attenuated level of a dehydratase/isomerase enzyme, such asan enzyme encoded by fabA. In other embodiments, the host cell isgenetically engineered to express an attenuated level of a ketoacyl-ACPsynthase, such as an enzyme encoded by fabB. In yet other embodiments,the host cell is genetically engineered to express a modified level of agene encoding a desaturase enzyme, such as desA.

In some embodiments, the polypeptide is from a bacterium, a plant, aninsect, a yeast, a fungus, or a mammal.

In certain embodiments, the polypeptide is from a bacterium. In someembodiments, the bacterium is a mycobacterium selected from the groupconsisting of Mycobacterium smegmatis, Mycobacterium abscessus,Mycobacterium avium, Mycobacterium bovis, Mycobacterium tuberculosis,Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium ulcerans.In other embodiments, the bacterium is Nocardia sp. NRRL 5646, Nocardiafarcinica, Streptomyces griseus, Salinispora arenicola, or Clavibactermichiganenesis.

In some embodiments, the method further includes culturing the host cellin the presence of at least one biological substrate for the fattyalcohol biosynthetic polypeptide.

In another aspect, the invention features a method of producing a fattyalcohol. The method includes expressing in a host cell a gene encoding afatty alcohol biosynthetic polypeptide comprising an amino acid sequencehaving at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity to the amino acid sequence of SEQ ID NO:94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, or 194. In some embodiments, the aminoacid sequence is SEQ ID NO:94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, or 194.

In some embodiments, the method further includes isolating the fattyalcohol from the host cell. In some embodiments, the fatty alcohol ispresent in the extracellular environment. In certain embodiments, thefatty alcohol is isolated from the extracellular environment of the hostcell. In some embodiments, the fatty alcohol is secreted from the hostcell. In alternative embodiments, the fatty alcohol is transported intothe extracellular environment. In other embodiments, the fatty alcoholis passively transported into the extracellular environment.

In some embodiments, the method further includes modifying theexpression of a gene encoding a fatty acid synthase in the host cell. Incertain embodiments, modifying the expression of a gene encoding a fattyacid synthase includes expressing a gene encoding a fatty acid synthasein the host cell and/or increasing the expression or activity of anendogenous fatty acid synthase in the host cell. In alternateembodiments, modifying the expression of a gene encoding a fatty acidsynthase includes attenuating a gene encoding a fatty acid synthase inthe host cell and/or decreasing the expression or activity of anendogenous fatty acid synthase in the host cell. In some embodiments,the fatty acid synthase is a thioesterase. In particular embodiments,the thioesterase is encoded by tesA, tesA without leader sequence, tesB,fatB, fatB2, fatB3, fatA, or fatA1.

In some embodiments, the method further includes expressing a geneencoding a fatty aldehyde biosynthetic polypeptide in the host cell. Inparticular embodiments, the fatty aldehyde biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 264, 266, 268,270, or 272, or a variant thereof.

In other embodiments, the host cell is genetically engineered to expressan attenuated level of a fatty acid degradation enzyme relative to awild type host cell. In some embodiments, the host cell is geneticallyengineered to express an attenuated level of an acyl-CoA synthaserelative to a wild type host cell. In particular embodiments, the hostcell expresses an attenuated level of an acyl-CoA synthase encoded byfadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.In certain embodiments, the genetically engineered host cell comprises aknockout of one or more genes encoding a fatty acid degradation enzyme,such as the aforementioned acyl-CoA synthase genes.

In yet other embodiments, the host cell is genetically engineered toexpress an attenuated level of a dehydratase/isomerase enzyme, such asan enzyme encoded by fabA or by a gene listed in FIG. 15. In someembodiments, the host cell comprises a knockout of fabA or a gene listedin FIG. 15. In other embodiments, the host cell is geneticallyengineered to express an attenuated level of a ketoacyl-ACP synthase,such as an enzyme encoded by fabB or by a gene listed in FIG. 16. Incertain embodiments, the host cell comprises a knockout of fabB or agene listed in FIG. 16. In yet other embodiments, the host cell isgenetically engineered to express a modified level of a gene encoding adesaturase enzyme, such as desA.

In some embodiments, the polypeptide is from a bacterium, a plant, aninsect, a yeast, a fungus, or a mammal.

In certain embodiments, the polypeptide is from a mammalian cell, plantcell, insect cell, yeast cell, fungus cell, filamentous fungi cell,bacterial cell, or any other organism described herein. In someembodiments, the bacterium is a mycobacterium selected from the groupconsisting of Mycobacterium smegmatis, Mycobacterium abscessus,Mycobacterium avium, Mycobacterium bovis, Mycobacterium tuberculosis,Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium ulcerans.In other embodiments, the bacterium is Nocardia sp. NRRL 5646, Nocardiafarcinica, Streptomyces griseus, Salinispora arenicola, or Clavibactermichiganenesis.

In some embodiments, the method further includes culturing the host cellin the presence of at least one biological substrate for the fattyalcohol biosynthetic polypeptide.

In another aspect, the invention features a method of producing a fattyalcohol. The method includes expressing in a host cell a polynucleotidethat hybridizes to a complement of the nucleotide sequence of SEQ IDNO:93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 187, 189, 191, or 193, or to a fragmentthereof, wherein the polynucleotide encodes a polypeptide having alcoholdehydrogenase activity.

In some embodiments, the method further includes isolating the fattyalcohol from the host cell. In some embodiments, the fatty alcohol ispresent in the extracellular environment. In certain embodiments, thefatty alcohol is isolated from the extracellular environment of the hostcell. In some embodiments, the fatty alcohol is secreted from the hostcell. In alternative embodiments, the fatty alcohol is transported intothe extracellular environment. In other embodiments, the fatty alcoholis passively transported into the extracellular environment.

In some embodiments, the polynucleotide hybridizes under low stringency,medium stringency, high stringency, or very high stringency conditions,to a complement of the nucleotide sequence of SEQ ID NO:93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, 191, or 193, or to a fragment thereof.

In some embodiments, the method further includes modifying theexpression of a gene encoding a fatty acid synthase in the host cell. Incertain embodiments, modifying the expression of a gene encoding a fattyacid synthase includes expressing a gene encoding a fatty acid synthasein the host cell and/or increasing the expression or activity of anendogenous fatty acid synthase in the host cell. In alternateembodiments, modifying the expression of a gene encoding a fatty acidsynthase includes attenuating a gene encoding a fatty acid synthase inthe host cell and/or decreasing the expression or activity of anendogenous fatty acid synthase in the host cell. In some embodiments,the fatty acid synthase is a thioesterase. In particular embodiments,the thioesterase is encoded by tesA, tesA without leader sequence, tesB,fatB, fatB2, fatB3, fatA, or fatA1.

In some embodiments, the method further includes expressing a geneencoding a fatty aldehyde biosynthetic polypeptide in the host cell. Inparticular embodiments, the fatty aldehyde biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 264, 266, 268,270, or 272, or a variant thereof.

In other embodiments, the host cell is genetically engineered to expressan attenuated level of a fatty acid degradation enzyme relative to awild type host cell. In some embodiments, the host cell is geneticallyengineered to express an attenuated level of an acyl-CoA synthaserelative to a wild type host cell. In particular embodiments, the hostcell expresses an attenuated level of an acyl-CoA synthase encoded byfadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.In certain embodiments, the genetically engineered host cell comprises aknockout of one or more genes encoding a fatty acid degradation enzyme,such as the aforementioned acyl-CoA synthase genes.

In yet other embodiments, the host cell is genetically engineered toexpress an attenuated level of a dehydratase/isomerase enzyme, such asan enzyme encoded by fabA or by a gene listed in FIG. 15. In someembodiments, the host cell comprises a knockout of fabA or a gene listedin FIG. 15. In other embodiments, the host cell is geneticallyengineered to express an attenuated level of a ketoacyl-ACP synthase,such as an enzyme encoded by fabB or by a gene listed in FIG. 16. Incertain embodiments, the host cell comprises a knockout of fabB or agene listed in FIG. 16. In yet other embodiments, the host cell isgenetically engineered to express a modified level of a gene encoding adesaturase enzyme, such as desA.

In some embodiments, the polynucleotide is from a bacterium, a plant, aninsect, a yeast, a fungus, or a mammal.

In certain embodiments, the polypeptide is from a mammalian cell, plantcell, insect cell, yeast cell, fungus cell, filamentous fungi cell,bacterial cell, or any other organism described herein. In someembodiments, the bacterium is a mycobacterium selected from the groupconsisting of Mycobacterium smegmatis, Mycobacterium abscessus,Mycobacterium avium, Mycobacterium bovis, Mycobacterium tuberculosis,Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium ulcerans.In other embodiments, the bacterium is Nocardia sp. NRRL 5646, Nocardiafarcinica, Streptomyces griseus, Salinispora arenicola, or Clavibactermichiganenesis.

In some embodiments, the method further includes culturing the host cellin the presence of at least one biological substrate for the fattyaldehyde biosynthetic polypeptide.

In another aspect, the invention features a method of producing a fattyalcohol. The method includes expressing in a host cell a recombinantvector comprising a fatty alcohol biosynthetic nucleotide sequencehaving at least about 70% sequence identity to the nucleotide sequenceof SEQ ID NO:93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143,145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171,173, 175, 177, 179, 181, 183, 185, 187, 189, 191, or 193. In someembodiments, the nucleotide sequence has at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99% sequence identity to the nucleotide sequence ofSEQ ID NO:93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, or 193. In someembodiments, the nucleotide sequence is of SEQ ID NO:93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, 191, or 193.

In some embodiments, the method further includes isolating the fattyalcohol from the host cell. In some embodiments, the fatty alcohol ispresent in the extracellular environment. In certain embodiments, thefatty alcohol is isolated from the extracellular environment of the hostcell. In some embodiments, the fatty alcohol is secreted from the hostcell. In alternative embodiments, the fatty alcohol is transported intothe extracellular environment. In other embodiments, the fatty alcoholis passively transported into the extracellular environment.

In some embodiments, the recombinant vector further comprises a promoteroperably linked to the nucleotide sequence. In certain embodiments, thepromoter is a developmentally-regulated, an organelle-specific, atissue-specific, an inducible, a constitutive, or a cell-specificpromoter.

In other embodiments, the recombinant vector comprises at least onesequence selected from the group consisting of (a) a regulatory sequenceoperatively coupled to the nucleotide sequence; (b) a selection markeroperatively coupled to the nucleotide sequence; (c) a marker sequenceoperatively coupled to the nucleotide sequence; (d) a purificationmoiety operatively coupled to the nucleotide sequence; (e) a secretionsequence operatively coupled to the nucleotide sequence; and (f) atargeting sequence operatively coupled to the nucleotide sequence.

In some embodiments, the recombinant vector is a plasmid.

In some embodiments, the host cell expresses a polypeptide encoded bythe recombinant vector. In some embodiments, the nucleotide sequence isstably incorporated into the genomic DNA of the host cell, and theexpression of the nucleotide sequence is under the control of aregulated promoter region.

In some embodiments, the method further includes modifying theexpression of a gene encoding a fatty acid synthase in the host cell. Incertain embodiments, modifying the expression of a gene encoding a fattyacid synthase includes expressing a gene encoding a fatty acid synthasein the host cell and/or increasing the expression or activity of anendogenous fatty acid synthase in the host cell. In alternateembodiments, modifying the expression of a gene encoding a fatty acidsynthase includes attenuating a gene encoding a fatty acid synthase inthe host cell and/or decreasing the expression or activity of anendogenous fatty acid synthase in the host cell. In some embodiments,the fatty acid synthase is a thioesterase. In particular embodiments,the thioesterase is encoded by tesA, tesA without leader sequence, tesB,fatB, fatB2, fatB3, fatA, or fatA1.

In some embodiments, the method further includes expressing a geneencoding a fatty aldehyde biosynthetic polypeptide in the host cell. Inparticular embodiments, the fatty aldehyde biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 264, 266, 268,270, or 272, or a variant thereof.

In other embodiments, the host cell is genetically engineered to expressan attenuated level of a fatty acid degradation enzyme relative to awild type host cell. In some embodiments, the host cell is geneticallyengineered to express an attenuated level of an acyl-CoA synthaserelative to a wild type host cell. In particular embodiments, the hostcell expresses an attenuated level of an acyl-CoA synthase encoded byfadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.In certain embodiments, the genetically engineered host cell comprises aknockout of one or more genes encoding a fatty acid degradation enzyme,such as the aforementioned acyl-CoA synthase genes.

In yet other embodiments, the host cell is genetically engineered toexpress an attenuated level of a dehydratase/isomerase enzyme, such asan enzyme encoded by fabA or by a gene listed in FIG. 15. In someembodiments, the host cell comprises a knockout of fabA or a gene listedin FIG. 15. In other embodiments, the host cell is geneticallyengineered to express an attenuated level of a ketoacyl-ACP synthase,such as an enzyme encoded by fabB or by a gene listed in FIG. 16. Incertain embodiments, the host cell comprises a knockout of fabB or agene listed in FIG. 16. In yet other embodiments, the host cell isgenetically engineered to express a modified level of a gene encoding adesaturase enzyme, such as desA.

In some embodiments, the method further includes culturing the host cellin the presence of at least one biological substrate for a fatty alcoholbiosynthetic polypeptide.

In any of the aspects of the invention described herein, the host cellcan be selected from the group consisting of a mammalian cell, plantcell, insect cell, yeast cell, fungus cell, filamentous fungi cell, andbacterial cell.

In some embodiments, the host cell is a Gram-positive bacterial cell. Inother embodiments, the host cell is a Gram-negative bacterial cell.

In some embodiments, the host cell is selected from the genusEscherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas,Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor,Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete,Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas,Schizosaccharomyces, Yarrowia, or Streptomyces.

In certain embodiments, the host cell is a Bacillus lentus cell, aBacillus brevis cell, a Bacillus stearothermophilus cell, a Bacilluslicheniformis cell, a Bacillus alkalophilus cell, a Bacillus coagulanscell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillusthuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell,a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.

In other embodiments, the host cell is a Trichoderma koningii cell, aTrichoderma viride cell, a Trichoderma reesei cell, a Trichodermalongibrachiatum cell, an Aspergillus awamori cell, an Aspergillusfumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulanscell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicolainsolens cell, a Humicola lanuginose cell, a Rhodococcus opacus cell, aRhizomucor miehei cell, or a Mucor michei cell.

In yet other embodiments, the host cell is a Streptomyces lividans cellor a Streptomyces murinus cell.

In yet other embodiments, the host cell is an Actinomycetes cell.

In some embodiments, the host cell is a Saccharomyces cerevisiae cell.In some embodiments, the host cell is a Saccharomyces cerevisiae cell.

In particular embodiments, the host cell is a cell from an eukaryoticplant, algae, cyanolacterium, green-sulfur bacterium, green non-sulfurbacterium, purple sulfur bacterium, purple non-sulfur bacterium,extremophile, yeast, fungus, engineered organisms thereof, or asynthetic organism. In some embodiments, the host cell is lightdependent or fixes carbon. In some embodiments, the host cell is lightdependent or fixes carbon. In some embodiments, the host cell hasautotrophic activity. In some embodiments, the host cell hasphotoautotrophic activity, such as in the presence of light. In someembodiments, the host cell is heterotrophic or mixotrophic in theabsence of light. In certain embodiments, the host cell is a cell fromAvabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, Zea mays,Botryococcuse braunii, Chlamydomonas reinhardtii, Dunaliela salina,Synechococcus Sp. PCC 7002, Synechococcus Sp. PCC 7942, SynechocystisSp. PCC 6803, Thermosynechococcus elongates BP-1, Chlorobium tepidum,Chloroflexus auranticus, Chromatiumm vinosum, Rhodospirillum rubrum,Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridiumljungdahlii, Clostridiuthermocellum, Penicillium chrysogenum, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pseudomonas fluorescens, or Zymomonas mobilis.

In other embodiments, the host cell is a CHO cell, a COS cell, a VEROcell, a BHK cell, a HeLa cell, a Cv1 cell, an MDCK cell, a 293 cell, a3T3 cell, or a PC12 cell.

In yet other embodiments, the host cell is an E. coli cell. In certainembodiments, the E. coli cell is a strain B, a strain C, a strain K, ora strain W E. coli cell.

In another aspect, the invention features a method of producing a fattyalcohol. The method includes contacting a substrate with (i) a fattyalcohol biosynthetic polypeptide comprising the amino acid sequence ofSEQ ID NO:94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, or 194, or a variantthereof, or (ii) a fatty alcohol biosynthetic polypeptide encoded by anucleotide sequence having at least about 70% identity to the nucleotidesequence of SEQ ID NO:93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, or 193, or avariant thereof. In some embodiments, the method further includespurifying the fatty alcohol.

In some embodiments, the fatty alcohol biosynthetic polypeptidecomprises the amino acid sequence of SEQ ID NO:94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, or 194 with one or more amino acid substitutions,additions, insertions, or deletions, wherein the polypeptide has alcoholdehydrogenase activity.

In some embodiments, the polypeptide comprises one or more of thefollowing conservative amino acid substitutions: replacement of analiphatic amino acid, such as alanine, valine, leucine, and isoleucine,with another aliphatic amino acid; replacement of a serine with athreonine; replacement of a threonine with a serine; replacement of anacidic residue, such as aspartic acid and glutamic acid, with anotheracidic residue; replacement of a residue bearing an amide group, such asasparagine and glutamine, with another residue bearing an amide group;exchange of a basic residue, such as lysine and arginine, with anotherbasic residue; and replacement of an aromatic residue, such asphenylalanine and tyrosine, with another aromatic residue. In someembodiments, the polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acidsubstitutions, additions, insertions, or deletions. In some embodiments,the polypeptide has alcohol dehydrogenase activity.

In some embodiments, the polypeptide has an amino acid sequence that isat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, or at least about 99% identical to theamino acid sequence of SEQ ID NO:94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, or194. In some embodiments, the polypeptide has the amino acid sequence ofSEQ ID NO:94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, or 194.

In some embodiments, the nucleotide sequence has at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% sequence identity to the nucleotide sequenceof SEQ ID NO:93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143,145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171,173, 175, 177, 179, 181, 183, 185, 187, 189, 191, or 193. In someembodiments, the nucleotide sequence is SEQ ID NO:93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, or 193.

In any of the aspects of the invention described herein, the methods canproduce fatty alcohols comprising a C₆-C₂₆ fatty alcohol. In someembodiments, the fatty alcohol comprises a C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, ora C₂₆ fatty alcohol. In particular embodiments, the fatty alcohol is aC₆, C₈, C₁₀, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, or C₁₈ fatty alcohol. Incertain embodiments, the hydroxyl group of the fatty alcohol is in theprimary (C₁) position.

In other embodiments, the fatty alcohol comprises a straight chain fattyalcohol. In other embodiments, the fatty alcohol comprises a branchedchain fatty alcohol. In yet other embodiments, the fatty alcoholcomprises a cyclic moiety.

In some embodiments, the fatty alcohol is an unsaturated fatty alcohol.In other embodiments, the fatty alcohol is a monounsaturated fattyalcohol. In certain embodiments, the unsaturated fatty alcohol is aC6:1, C7:1, C8:1, C9:1, C10:1, C11:1, C12:1, C13:1, C14:1, C15:1, C16:1,C17:1, C18:1, C19:1, C20:1, C21:1, C22:1, C23:1, C24:1, C25:1, or aC26:1 unsaturated fatty alcohol. In yet other embodiments, the fattyalcohol is unsaturated at the omega-7 position. In certain embodiments,the unsaturated fatty alcohol comprises a cis double bond.

In yet other embodiments, the fatty alcohol is a saturated fattyalcohol.

In any of the aspects of the invention described herein, a substrate fora fatty aldehyde biosynthetic polypeptide can be a fatty acid. In someembodiments, the fatty acid comprises a C₆-C₂₆ fatty acid. In someembodiments, the fatty acid comprises a C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂,C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, or aC₂₆ fatty acid. In particular embodiments, the fatty acid is a C₆, C₈,C₁₀, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, or C₁₈ fatty acid.

In other embodiments, the fatty acid comprises a straight chain fattyacid. In other embodiments, the fatty acid comprises a branched chainfatty acid. In yet other embodiments, the fatty acid comprises a cyclicmoiety.

In some embodiments, the fatty acid is an unsaturated fatty acid. Inother embodiments, the fatty acid is a monounsaturated fatty acid. Inyet other embodiments, the fatty acid is a saturated fatty acid.

In another aspect, the invention features a genetically engineeredmicroorganism comprising an exogenous control sequence stablyincorporated into the genomic DNA of the microorganism upstream of apolynucleotide comprising a nucleotide sequence having at least about70% sequence identity to the nucleotide sequence of SEQ ID NO:17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 187, 189, 191, 193, 263, 265, 267, 269, or 271,wherein the microorganism produces an increased level of a fatty alcoholrelative to a wild-type microorganism.

In some embodiments, the nucleotide sequence has at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% sequence identity to the nucleotide sequenceof SEQ ID NO:17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 263, 265,267, 269, or 271. In some embodiments, the nucleotide sequence is SEQ IDNO:17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 263, 265, 267, 269, or271.

In some embodiments, the polynucleotide is endogenous to themicroorganism.

In other embodiments, the microorganism is genetically engineered toexpress a modified level of a gene encoding a fatty acid synthase in thehost cell. In certain embodiments, the microorganism expresses arecombinant gene encoding a fatty acid synthase or expresses anincreased level of an endogenous fatty acid synthase. In alternateembodiments, the microorganism expresses an attenuated level of a geneencoding a fatty acid synthase in the host cell and/or a decreasedexpression or activity of an endogenous fatty acid synthase. In someembodiments, the fatty acid synthase is a thioesterase. In particularembodiments, the thioesterase is encoded by tesA, tesA without leadersequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.

In other embodiments, the microorganism is genetically engineered toexpress an attenuated level of a fatty acid degradation enzyme relativeto a wild type microorganism. In some embodiments, the microorganismexpresses an attenuated level of an acyl-CoA synthase relative to a wildtype microorganism. In particular embodiments, the microorganismexpresses an attenuated level of an acyl-CoA synthase encoded by fadD,fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.In certain embodiments, the microorganism comprises a knockout of one ormore genes encoding a fatty acid degradation enzyme, such as theaforementioned acyl-CoA synthase genes.

In yet other embodiments, the microorganism is genetically engineered toexpress an attenuated level of a dehydratase/isomerase enzyme, such asan enzyme encoded by fabA or by a gene listed in FIG. 15. In someembodiments, the microorganism comprises a knockout of fabA or a genelisted in FIG. 15. In other embodiments, the microorganism isgenetically engineered to express an attenuated level of a ketoacyl-ACPsynthase, such as an enzyme encoded by fabB or by a gene listed in FIG.16. In certain embodiments, the microorganism comprises a knockout offabB or a gene listed in FIG. 16. In yet other embodiments, themicroorganism is genetically engineered to express a modified level of agene encoding a desaturase enzyme, such as desA.

In some embodiments, the microorganism is a bacterium. In certainembodiments, the bacterium is a Gram-negative or a Gram-positivebacterium.

In some embodiments, the microorganism is a mycobacterium selected fromthe group consisting of Mycobacterium smegmatis, Mycobacteriumabscessus, Mycobacterium avium, Mycobacterium bovis, Mycobacteriumtuberculosis, Mycobacterium leprae, Mycobacterium marinum, andMycobacterium ulcerans.

In other embodiments, the microorganism is Nocardia sp. NRRL 5646,Nocardia farcinica, Streptomyces griseus, Salinispora arenicola, orClavibacter michiganenesis.

In another aspect, the invention features a fatty alcohol produced byany of the methods or any of the microorganisms described herein, or asurfactant comprising a fatty alcohol produced by any of the methods orany of the microorganisms described herein.

In some embodiments, the fatty alcohol has a δ¹³C of about −15.4 orgreater. In certain embodiments, the fatty alcohol has a δ¹³C of about−15.4 to about −10.9, or of about −13.92 to about −13.84.

In some embodiments, the fatty alcohol has an f_(M) ¹⁴C of at leastabout 1.003. In certain embodiments, the fatty alcohol has an f_(M) ¹⁴Cof at least about 1.01 or at least about 1.5. In some embodiments, thefatty alcohol has an f_(M) ¹⁴C of about 1.111 to about 1.124.

In any of the aspects described herein, a fatty alcohol is produced at ayield of about 25 mg/L, about 50 mg/L, about 75 mg/L, about 100 mg/L,about 125 mg/L, about 150 mg/L, about 175 mg/L, about 200 mg/L, about225 mg/L, about 250 mg/L, about 275 mg/L, about 300 mg/L, about 325mg/L, about 350 mg/L, about 375 mg/L, about 400 mg/L, about 425 mg/L,about 450 mg/L, about 475 mg/L, about 500 mg/L, about 525 mg/L, about550 mg/L, about 575 mg/L, about 600 mg/L, about 625 mg/L, about 650mg/L, about 675 mg/L, about 700 mg/L, about 725 mg/L, about 750 mg/L,about 775 mg/L, about 800 mg/L, about 825 mg/L, about 850 mg/L, about875 mg/L, about 900 mg/L, about 925 mg/L, about 950 mg/L, about 975mg/L, about 1000 g/L, about 1050 mg/L, about 1075 mg/L, about 1100 mg/L,about 1125 mg/L, about 1150 mg/L, about 1175 mg/L, about 1200 mg/L,about 1225 mg/L, about 1250 mg/L, about 1275 mg/L, about 1300 mg/L,about 1325 mg/L, about 1350 mg/L, about 1375 mg/L, about 1400 mg/L,about 1425 mg/L, about 1450 mg/L, about 1475 mg/L, about 1500 mg/L,about 1525 mg/L, about 1550 mg/L, about 1575 mg/L, about 1600 mg/L,about 1625 mg/L, about 1650 mg/L, about 1675 mg/L, about 1700 mg/L,about 1725 mg/L, about 1750 mg/L, about 1775 mg/L, about 1800 mg/L,about 1825 mg/L, about 1850 mg/L, about 1875 mg/L, about 1900 mg/L,about 1925 mg/L, about 1950 mg/L, about 1975 mg/L, about 2000 mg/L, ormore.

In another aspect, the invention features a method of making a fattyalcohol described herein. The method includes culturing a host celldescribed herein in a medium having a low level of iron, underconditions sufficient to produce a fatty alcohol, as described herein.In particular embodiments, the medium contains less than about 500 μMiron, less than about 400 μM iron, less than about 300 μM iron, lessthan about 200 μM iron, less than about 150 μM iron, less than about 100μM iron, less than about 90 μM iron, less than about 80 μM iron, lessthan about 70 μM iron, less than about 60 μM iron, less than about 50 μMiron, less than about 40 μM iron, less than about 30 μM iron, less thanabout 20 μM iron, less than about 10 μM iron, or less than about 5 μMiron. In certain embodiments, the medium does not contain iron.

In any of the aspects described herein, a fatty alcohol is produced in ahost cell or a microorganism described herein from a carbon source.

The following figures are presented for the purpose of illustrationonly, and are not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation of fatty alcohols produced byrecombinant E. coli strains transformed with various plasmids.

FIG. 2 is a graphic representation of two GC/MS traces of organiccompounds produced by recombinant E. coli strains transformed withvarious plasmids.

FIG. 3 is a schematic of a new pathway for fatty alcohol production.

FIG. 4 is a representation of a gel of PCR products from MG1655 wildtype cells, ΔfadD::cm cells, and ΔfadD cells.

FIG. 5A is a GC/MS trace of fatty alcohol production in MG1655(DE3,ΔfadD)/pETDUet-1-tesA+pHZ1.140B cells. FIG. 5B is a GC/MS trace of fattyalcohol production in MG16655(DE3, ΔfadD,yjgB::kan)/pETDUet-1-tesA+pHZ1.140B cells. FIG. 5C is a GC/MS trace offatty alcohol production in MG16655(DE3, ΔfadD,yjgB::kan)/pDF1+pHZ1.140B cells. The arrows in FIG. 5A, FIG. 5B, andFIG. 5C indicate the absence of C12:0 fatty aldehydes.

FIG. 6 is a listing of the nucleotide sequence and the correspondingamino acid sequence of Nocardia sp. NRRL 5646 car gene.

FIG. 7 is a listing of amino acid sequence motifs for CAR homologs.

FIG. 8 is a listing of nucleotide and amino acid sequences of carhomolog genes.

FIG. 9 is a table identifying exemplary genes that can be expressed,overexpressed, or attenuated to increase production of particularsubstrates.

FIG. 10 is a listing of nucleotide and amino acid sequences of alcoholdehydrogenase genes.

FIG. 11 is a graphic representation of fatty alcohol production invarious deletion mutants of E. coli.

FIG. 12 is a graphic representation of fatty alcohol production invarious deletion mutants of E. coli.

FIG. 13 is a GC/MS trace of saturated fatty alcohol production in E.coli.

FIG. 14A is a graphic representation of fatty alcohol production invarious Hu9 culture media. FIG. 14B is a graphic representation of fattyalcohol production in various Hu9 culture media.

FIG. 15 is a listing of nucleotide and amino acid sequences of fabArelated genes.

FIG. 16 is a listing of nucleotide and amino acid sequences of fabBrelated genes.

FIG. 17 is a listing of additional nucleotide and amino acid sequencesof the disclosure.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein, including GenBankdatabase sequences, are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DEFINITIONS

Throughout the specification, a reference may be made using anabbreviated gene name or polypeptide name, but it is understood thatsuch an abbreviated gene or polypeptide name represents the genus ofgenes or polypeptides. Such gene names include all genes encoding thesame polypeptide and homologous polypeptides having the samephysiological function. Polypeptide names include all polypeptides thathave the same activity (e.g., that catalyze the same fundamentalchemical reaction).

Unless otherwise indicated, the accession numbers referenced herein arederived from the NCBI database (National Center for BiotechnologyInformation) maintained by the National Institute of Health, U.S.A.Unless otherwise indicated, the accession numbers are as provided in thedatabase as of October 2008.

EC numbers are established by the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology (NC-IUBMB)which is accessible through the Internet. The EC numbers referencedherein are derived from the KEGG Ligand database, maintained by theKyoto Encyclopedia of Genes and Genomics, sponsored in part by theUniversity of Tokyo. Unless otherwise indicated, the EC numbers are asprovided in the database as of October 2008.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” is used herein to mean a value±20% of a given numericalvalue. Thus, “about 60%” means a value of between 60±(20% of 60) (i.e.,between 48 and 70).

As used herein, the term “alcohol dehydrogenase” (EC 1.1.1.*) is apeptide capable of catalyzing the conversion of a fatty aldehyde to analcohol (e.g., fatty alcohol). Additionally, one of ordinary skill inthe art will appreciate that some alcohol dehydrogenases will catalyzeother reactions as well. For example, some alcohol dehydrogenases willaccept other substrates in addition to fatty aldehydes. Suchnon-specific alcohol dehydrogenases are, therefore, also included inthis definition. Nucleic acid sequences encoding alcohol dehydrogenasesare known in the art, and such alcohol dehydrogenases are publiclyavailable. Exemplary GenBank Accession Numbers are provided in FIG. 9.

As used herein, the term “attenuate” means to weaken, reduce ordiminish. For example, a polypeptide can be attenuated by modifying thepolypeptide to reduce its activity (e.g., by modifying a nucleotidesequence that encodes the polypeptide).

As used herein, the term “biodiesel” means a biofuel that can be asubstitute of diesel, which is derived from petroleum. Biodiesel can beused in internal combustion diesel engines in either a pure form, whichis referred to as “neat” biodiesel, or as a mixture in any concentrationwith petroleum-based diesel. Biodiesel can include esters orhydrocarbons, such as alcohols.

As used therein, the term “biofuel” refers to any fuel derived frombiomass. Biofuels can be substituted for petroleum based fuels. Forexample, biofuels are inclusive of transportation fuels (e.g., gasoline,diesel, jet fuel, etc.), heating fuels, and electricity-generatingfuels. Biofuels are a renewable energy source.

As used herein, the term “biomass” refers to any biological materialfrom which a carbon source is derived. In some instances, a biomass isprocessed into a carbon source, which is suitable for bioconversion. Inother instances, the biomass may not require further processing into acarbon source. The carbon source can be converted into a biofuel. Oneexemplary source of biomass is plant matter or vegetation. For example,corn, sugar cane, or switchgrass can be used as biomass. Anothernon-limiting example of biomass is metabolic wastes, such as animalmatter, for example cow manure. In addition, biomass may include algaeand other marine plants. Biomass also includes waste products fromindustry, agriculture, forestry, and households. Examples of such wasteproducts that can be used as biomass are fermentation waste, ensilage,straw, lumber, sewage, garbage, cellulosic urban waste, and foodleftovers. Biomass also includes sources of carbon, such ascarbohydrates (e.g., monosaccharides, disaccharides, orpolysaccharides).

As used herein, the phrase “carbon source” refers to a substrate orcompound suitable to be used as a source of carbon for prokaryotic orsimple eukaryotic cell growth. Carbon sources can be in various forms,including, but not limited to polymers, carbohydrates, acids, alcohols,aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO₂).These include, for example, various monosaccharides, such as glucose,fructose, mannose, and galactose; oligosaccharides, such asfructo-oligosaccharide and galacto-oligosaccharide; polysaccharides suchas xylose and arabinose; disaccharides, such as sucrose, maltose, andturanose; cellulosic material, such as methyl cellulose and sodiumcarboxymethyl cellulose; saturated or unsaturated fatty acid esters,such as succinate, lactate, and acetate; alcohols, such as ethanol,methanol, and glycerol, or mixtures thereof. The carbon source can alsobe a product of photosynthesis, including, but not limited to, glucose.A preferred carbon source is biomass. Another preferred carbon source isglucose.

A nucleotide sequence is “complementary” to another nucleotide sequenceif each of the bases of the two sequences matches (i.e., is capable offorming Watson Crick base pairs). The term “complementary strand” isused herein interchangeably with the term “complement”. The complementof a nucleic acid strand can be the complement of a coding strand or thecomplement of a non-coding strand.

As used herein, a “cloud point lowering additive” is an additive addedto a composition to decrease or lower the cloud point of a solution.

As used herein, the phrase “cloud point of a fluid” means thetemperature at which dissolved solids are no longer completely soluble.Below this temperature, solids begin precipitating as a second phasegiving the fluid a cloudy appearance. In the petroleum industry, cloudpoint refers to the temperature below which a solidified material orother heavy hydrocarbon crystallizes in a crude oil, refined oil, orfuel to form a cloudy appearance. The presence of solidified materialsinfluences the flowing behavior of the fluid, the tendency of the fluidto clog fuel filters, injectors, etc., the accumulation of solidifiedmaterials on cold surfaces (e.g., a pipeline or heat exchanger fouling),and the emulsion characteristics of the fluid with water.

As used herein, the term “conditions sufficient to allow expression”means any conditions that allow a host cell to produce a desiredproduct, such as a polypeptide or fatty aldehyde described herein.Suitable conditions include, for example, fermentation conditions.Fermentation conditions can comprise many parameters, such astemperature ranges, levels of aeration, and media composition. Each ofthese conditions, individually and in combination, allows the host cellto grow. Exemplary culture media include broths or gels. Generally, themedium includes a carbon source, such as glucose, fructose, cellulose,or the like, that can be metabolized by a host cell directly. Inaddition, enzymes can be used in the medium to facilitate themobilization (e.g., the depolymerization of starch or cellulose tofermentable sugars) and subsequent metabolism of the carbon source.

To determine if conditions are sufficient to allow expression, a hostcell can be cultured, for example, for about 4, 8, 12, 24, 36, or 48hours. During and/or after culturing, samples can be obtained andanalyzed to determine if the conditions allow expression. For example,the host cells in the sample or the medium in which the host cells weregrown can be tested for the presence of a desired product. When testingfor the presence of a product, assays, such as, but not limited to, thinlayer chromatography (“TLC”), high pressure liquid chromatography(“HPLC”), gas chromatography with flame ionization detection (“GC/FID”),gas chromatograph-mass spectrometry (“GC/MS”), liquidchromatography-mass spectrometry (“LC/MS”), mass spectrometry (“MS”),can be used.

It is understood that the polypeptides described herein may haveadditional conservative or non-essential amino acid substitutions, whichdo not have a substantial effect on the polypeptide functions. Whetheror not a particular substitution will be tolerated (i.e., will notadversely affect desired biological properties, such as carboxylic acidreductase activity) can be determined as described in Bowie et al.Science (1990) 247:1306 1310. A “conservative amino acid substitution”is one in which the amino acid residue is replaced with an amino acidresidue having a similar side chain. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine), and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

As used herein, “control element” means a transcriptional controlelement. Control elements include promoters and enhancers. The term“promoter element,” “promoter,” or “promoter sequence” refers to a DNAsequence that functions as a switch that activates the expression of agene. If the gene is activated, it is said to be transcribed orparticipating in transcription. Transcription involves the synthesis ofmRNA from the gene. A promoter, therefore, serves as a transcriptionalregulatory element and also provides a site for initiation oftranscription of the gene into mRNA. Control elements interactspecifically with cellular proteins involved in transcription (Maniatiset al., Science 236:1237, 1987).

As used herein, the term “fatty acid” means a carboxylic acid having theformula RCOOH. R represents an aliphatic group, preferably an alkylgroup. R can comprise between about 4 and about 22 carbon atoms. Fattyacids can be saturated, monounsaturated, or polyunsaturated. In apreferred embodiment, the fatty acid is made from a fatty acidbiosynthetic pathway.

As used herein, the term “fatty acid biosynthetic pathway” means abiosynthetic pathway that produces fatty acids. The fatty acidbiosynthetic pathway includes fatty acid synthases that can beengineered, as described herein, to produce fatty acids, and in someembodiments can be expressed with additional enzymes to produce fattyacids having desired carbon chain characteristics.

As used herein, the term “fatty acid degradation enzyme” means an enzymeinvolved in the breakdown or conversion of a fatty acid or fatty acidderivative into another product. A nonlimiting example of a fatty aciddegradation enzyme is an acyl-CoA synthase. Additional examples of fattyacid degradation enzymes are described herein.

As used herein, the term “fatty acid derivative” means products made inpart from the fatty acid biosynthetic pathway of the production hostorganism. “Fatty acid derivative” also includes products made in partfrom acyl-ACP or acyl-ACP derivatives. The fatty acid biosyntheticpathway includes fatty acid synthase enzymes which can be engineered asdescribed herein to produce fatty acid derivatives, and in some examplescan be expressed with additional enzymes to produce fatty acidderivatives having desired carbon chain characteristics. Exemplary fattyacid derivatives include for example, fatty acids, acyl-CoA, fattyaldehyde, short and long chain alcohols, hydrocarbons, fatty alcohols,and esters (e.g., waxes, fatty acid esters, or fatty esters).

As used herein, the term “fatty acid derivative enzyme” means any enzymethat may be expressed or overexpressed in the production of fatty acidderivatives. These enzymes may be part of the fatty acid biosyntheticpathway. Non-limiting examples of fatty acid derivative enzymes includefatty acid synthases, thioesterases, acyl-CoA synthases, acyl-CoAreductases, alcohol dehydrogenases, alcohol acyltransferases, fattyalcohol-forming acyl-CoA reductases, fatty acid (carboxylic acid)reductases, acyl-ACP reductases, fatty acid hydroxylases, acyl-CoAdesaturases, acyl-ACP desaturases, acyl-CoA oxidases, acyl-CoAdehydrogenases, ester synthases, and alkane biosynthetic polypeptides,etc. Fatty acid derivative enzymes can convert a substrate into a fattyacid derivative. In some examples, the substrate may be a fatty acidderivative that the fatty acid derivative enzyme converts into adifferent fatty acid derivative.

As used herein, “fatty acid enzyme” means any enzyme involved in fattyacid biosynthesis. Fatty acid enzymes can be modified in host cells toproduce fatty acids. Non-limiting examples of fatty acid enzymes includefatty acid synthases and thioesterases. Additional examples of fattyacid enzymes are described herein.

As used herein, “fatty acid synthase” means any enzyme involved in fattyacid biosynthesis. Fatty acid synthases can be expressed oroverexpressed in host cells to produce fatty acids. A non-limitingexample of a fatty acid synthase is a thioesterase. Additional examplesof fatty acid synthases are described herein.

As used herein, “fatty aldehyde” means an aldehyde having the formulaRCHO characterized by an unsaturated carbonyl group (C═O). In apreferred embodiment, the fatty aldehyde is any aldehyde made from afatty acid or fatty acid derivative. In one embodiment, the R group isat least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 carbons in length.

R can be straight or branched chain. The branched chains may have one ormore points of branching. In addition, the branched chains may includecyclic branches.

Furthermore, R can be saturated or unsaturated. If unsaturated, the Rcan have one or more points of unsaturation.

In one embodiment, the fatty aldehyde is produced biosynthetically.

Fatty aldehydes have many uses. For example, fatty aldehydes can be usedto produce many specialty chemicals. For example, fatty aldehydes areused to produce polymers, resins, dyes, flavorings, plasticizers,perfumes, pharmaceuticals, and other chemicals. Some are used assolvents, preservatives, or disinfectants. Some natural and syntheticcompounds, such as vitamins and hormones, are aldehydes.

The terms “fatty aldehyde biosynthetic polypeptide”, “carboxylic acidreductase”, and “CAR” are used interchangeably herein.

As used herein, “fatty alcohol” means an alcohol having the formula ROH.In a preferred embodiment, the fatty alcohol is any alcohol made from afatty acid or fatty acid derivative. In one embodiment, the R group isat least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 carbons in length.

R can be straight or branched chain. The branched chains may have one ormore points of branching. In addition, the branched chains may includecyclic branches.

Furthermore, R can be saturated or unsaturated. If unsaturated, the Rcan have one or more points of unsaturation.

In one embodiment, the fatty alcohol is produced biosynthetically.

Fatty alcohols have many uses. For example, fatty alcohols can be usedto produce many specialty chemicals. For example, fatty alcohols areused as a biofuel; as solvents for fats, waxes, gums, and resins; inpharmaceutical salves, emolients and lotions; as lubricating-oiladditives; in detergents and emulsifiers; as textile antistatic andfinishing agents; as plasticizers; as nonionic surfactants; and incosmetics, for examples as thickeners.

As used herein, “fraction of modern carbon” or “f_(M)” has the samemeaning as defined by National Institute of Standards and Technology(NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known asoxalic acids standards HOxI and HOxII, respectively. The fundamentaldefinition relates to 0.95 times the ¹⁴C/¹²C isotope ratio HOxI(referenced to AD 1950). This is roughly equivalent to decay-correctedpre-Industrial Revolution wood. For the current living biosphere (plantmaterial), f_(M) is approximately 1.1.

“Gene knockout”, as used herein, refers to a procedure by which a geneencoding a target protein is modified or inactivated so to reduce oreliminate the function of the intact protein. Inactivation of the genemay be performed by general methods such as mutagenesis by UVirradiation or treatment with N-methyl-N′-nitro-N-nitrosoguanidine,site-directed mutagenesis, homologous recombination, insertion-deletionmutagenesis, or “Red-driven integration” (Datsenko et al., Proc. Natl.Acad. Sci. USA, 97:6640-45, 2000). For example, in one embodiment, aconstruct is introduced into a host cell, such that it is possible toselect for homologous recombination events in the host cell. One ofskill in the art can readily design a knock-out construct including bothpositive and negative selection genes for efficiently selectingtransfected cells that undergo a homologous recombination event with theconstruct. The alteration in the host cell may be obtained, for example,by replacing through a single or double crossover recombination a wildtype DNA sequence by a DNA sequence containing the alteration. Forconvenient selection of transformants, the alteration may, for example,be a DNA sequence encoding an antibiotic resistance marker or a genecomplementing a possible auxotrophy of the host cell. Mutations include,but are not limited to, deletion-insertion mutations. An example of suchan alteration includes a gene disruption, i.e., a perturbation of a genesuch that the product that is normally produced from this gene is notproduced in a functional form. This could be due to a complete deletion,a deletion and insertion of a selective marker, an insertion of aselective marker, a frameshift mutation, an in-frame deletion, or apoint mutation that leads to premature termination. In some instances,the entire mRNA for the gene is absent. In other situations, the amountof mRNA produced varies.

Calculations of “homology” between two sequences can be performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence that isaligned for comparison purposes is at least about 30%, preferably atleast about 40%, more preferably at least about 50%, even morepreferably at least about 60%, and even more preferably at least about70%, at least about 80%, at least about 90%, or about 100% of the lengthof the reference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein, amino acid or nucleic acid “identity” is equivalent toamino acid or nucleic acid “homology”). The percent identity between thetwo sequences is a function of the number of identical positions sharedby the sequences, taking into account the number of gaps and the lengthof each gap, which need to be introduced for optimal alignment of thetwo sequences.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent homology between twoamino acid sequences is determined using the Needleman and Wunsch(1970), J. Mol. Biol. 48:444 453, algorithm that has been incorporatedinto the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent homology between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularlypreferred set of parameters (and the one that should be used if thepractitioner is uncertain about which parameters should be applied todetermine if a molecule is within a homology limitation of the claims)are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extendpenalty of 4, and a frameshift gap penalty of 5.

As used herein, a “host cell” is a cell used to produce a productdescribed herein (e.g., a fatty alcohol described herein). A host cellcan be modified to express or overexpress selected genes or to haveattenuated expression of selected genes. Non-limiting examples of hostcells include plant, animal, human, bacteria, yeast, or filamentousfungi cells.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueousand nonaqueous methods are described in that reference and either methodcan be used. Specific hybridization conditions referred to herein are asfollows: 1) low stringency hybridization conditions in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by two washes in0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes canbe increased to 55° C. for low stringency conditions); 2) mediumstringency hybridization conditions in 6×SSC at about 45° C., followedby one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringencyhybridization conditions in 6×SSC at about 45° C., followed by one ormore washes in 0.2.×SSC, 0.1% SDS at 65° C.; and preferably 4) very highstringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Veryhigh stringency conditions (4) are the preferred conditions unlessotherwise specified.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs or RNAs,respectively, that are present in the natural source of the nucleicacid. Moreover, by an “isolated nucleic acid” is meant to includenucleic acid fragments, which are not naturally occurring as fragmentsand would not be found in the natural state. The term “isolated” is alsoused herein to refer to polypeptides, which are isolated from othercellular proteins and is meant to encompass both purified andrecombinant polypeptides. The term “isolated” as used herein also refersto a nucleic acid or peptide that is substantially free of cellularmaterial, viral material, or culture medium when produced by recombinantDNA techniques. The term “isolated” as used herein also refers to anucleic acid or peptide that is substantially free of chemicalprecursors or other chemicals when chemically synthesized. The term“isolated”, as used herein with respect to products, such as fattyalcohols, refers to products that are isolated from cellular components,cell culture media, or chemical or synthetic precursors.

As used herein, the “level of expression of a gene in a cell” refers tothe level of mRNA, pre-mRNA nascent transcript(s), transcript processingintermediates, mature mRNA(s), and degradation products encoded by thegene in the cell.

As used herein, the term “microorganism” means prokaryotic andeukaryotic microbial species from the domains Archaea, Bacteria andEucarya, the latter including yeast and filamentous fungi, protozoa,algae, or higher Protista. The terms “microbial cells” (i.e., cells frommicrobes) and “microbes” are used interchangeably and refer to cells orsmall organisms that can only be seen with the aid of a microscope.

As used herein, the term “nucleic acid” refers to polynucleotides, suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides, ESTs, chromosomes,cDNAs, mRNAs, and rRNAs.

As used herein, the term “operably linked” means that selectednucleotide sequence (e.g., encoding a polypeptide described herein) isin proximity with a promoter to allow the promoter to regulateexpression of the selected DNA. In addition, the promoter is locatedupstream of the selected nucleotide sequence in terms of the directionof transcription and translation. By “operably linked” is meant that anucleotide sequence and a regulatory sequence(s) are connected in such away as to permit gene expression when the appropriate molecules (e.g.,transcriptional activator proteins) are bound to the regulatorysequence(s).

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, “overexpress” means to express or cause to be expresseda nucleic acid, polypeptide, or hydrocarbon in a cell at a greaterconcentration than is normally expressed in a corresponding wild-typecell. For example, a polypeptide can be “overexpressed” in a recombinanthost cell when the polypeptide is present in a greater concentration inthe recombinant host cell compared to its concentration in anon-recombinant host cell of the same species.

As used herein, “partition coefficient” or “P,” is defined as theequilibrium concentration of a compound in an organic phase divided bythe concentration at equilibrium in an aqueous phase (e.g., fermentationbroth). In one embodiment of a bi-phasic system described herein, theorganic phase is formed by the fatty aldehyde during the productionprocess. However, in some examples, an organic phase can be provided,such as by providing a layer of octane, to facilitate productseparation. When describing a two phase system, the partitioncharacteristics of a compound can be described as logP. For example, acompound with a logP of 1 would partition 10:1 to the organic phase. Acompound with a logP of −1 would partition 1:10 to the organic phase. Bychoosing an appropriate fermentation broth and organic phase, a fattyaldehyde with a high logP value can separate into the organic phase evenat very low concentrations in the fermentation vessel.

As used herein, the term “purify,” “purified,” or “purification” meansthe removal or isolation of a molecule from its environment by, forexample, isolation or separation. “Substantially purified” molecules areat least about 60% free, preferably at least about 75% free, and morepreferably at least about 90% free from other components with which theyare associated. As used herein, these terms also refer to the removal ofcontaminants from a sample. For example, the removal of contaminants canresult in an increase in the percentage of fatty alcohol in a sample.For example, when fatty alcohols are produced in a host cell, the fattyalcohols can be purified by the removal of host cell proteins. Afterpurification, the percentage of fatty alcohols in the sample isincreased.

The terms “purify,” “purified,” and “purification” do not requireabsolute purity. They are relative terms. Thus, for example, when fattyalcohols are produced in host cells, a purified fatty alcohol is onethat is substantially separated from other cellular components (e.g.,nucleic acids, polypeptides, lipids, carbohydrates, or otherhydrocarbons). In another example, a purified fatty alcohol preparationis one in which the fatty alcohol is substantially free fromcontaminants, such as those that might be present followingfermentation. In some embodiments, a fatty alcohol is purified when atleast about 50% by weight of a sample is composed of the fatty alcohol.In other embodiments, a fatty alcohol is purified when at least about60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% or more by weight of asample is composed of the fatty alcohol.

As used herein, the term “recombinant polypeptide” refers to apolypeptide that is produced by recombinant DNA techniques, whereingenerally DNA encoding the expressed protein or RNA is inserted into asuitable expression vector and that is in turn used to transform a hostcell to produce the polypeptide or RNA.

As used herein, the term “substantially identical” (or “substantiallyhomologous”) is used to refer to a first amino acid or nucleotidesequence that contains a sufficient number of identical or equivalent(e.g., with a similar side chain, e.g., conserved amino acidsubstitutions) amino acid residues or nucleotides to a second amino acidor nucleotide sequence such that the first and second amino acid ornucleotide sequences have similar activities.

As used herein, the term “synthase” means an enzyme which catalyzes asynthesis process. As used herein, the term synthase includes synthases,synthetases, and ligases.

As used herein, the term “transfection” means the introduction of anucleic acid (e.g., via an expression vector) into a recipient cell bynucleic acid-mediated gene transfer.

As used herein, “transformation” refers to a process in which a cell'sgenotype is changed as a result of the cellular uptake of exogenous DNAor RNA. This may result in the transformed cell expressing a recombinantform of an RNA or polypeptide. In the case of antisense expression fromthe transferred gene, the expression of a naturally-occurring form ofthe polypeptide is disrupted.

As used herein, a “transport protein” is a polypeptide that facilitatesthe movement of one or more compounds in and/or out of a cellularorganelle and/or a cell.

As used herein, a “variant” of polypeptide X refers to a polypeptidehaving the amino acid sequence of peptide X in which one or more aminoacid residues is altered. The variant may have conservative changes ornonconservative changes. Guidance in determining which amino acidresidues may be substituted, inserted, or deleted without affectingbiological activity may be found using computer programs well known inthe art, for example, LASERGENE software (DNASTAR).

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to that of agene or the coding sequence thereof. This definition may also include,for example, “allelic,” “splice,” “species,” or “polymorphic” variants.A splice variant may have significant identity to a referencepolynucleotide, but will generally have a greater or fewer number ofpolynucleotides due to alternative splicing of exons during mRNAprocessing. The corresponding polypeptide may possess additionalfunctional domains or an absence of domains. Species variants arepolynucleotide sequences that vary from one species to another. Theresulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of useful vector is an episome (i.e., a nucleic acidcapable of extra-chromosomal replication). Useful vectors are thosecapable of autonomous replication and/or expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids,” whichrefer generally to circular double stranded DNA loops that, in theirvector form, are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably, as theplasmid is the most commonly used form of vector. However, also includedare such other forms of expression vectors that serve equivalentfunctions and that become known in the art subsequently hereto.

The invention is based, at least in part, on the discovery of a newpathway for fatty alcohol biosynthesis in E. coli that utilize, in part,genes that encode fatty aldehyde biosynthetic polypeptides. The fattyalcohols can be produced by a biosynthetic pathway depicted in FIG. 3.In this pathway, a fatty acid is first activated by ATP and then reducedby a carboxylic acid reductase (CAR)-like enzyme to generate a fattyaldehyde. The fatty aldehyde can then be further reduced into a fattyalcohol by an alcohol dehydrogenase(s), such as alrAadp1 or yjgB. Asdemonstrated herein, yjgB may be the presumed alcohol dehydrogenase,whose substrates includes fatty aldehydes, for example fatty aldehydeswith carbon chain lengths from C₁₀ to C₁₈.

Fatty Aldehyde Biosynthetic Genes, Fatty Alcohol Biosynthetic Genes, andVariants

The methods described herein can be used to produce fatty alcohols, forexample, from fatty aldehydes. In some instances, a fatty aldehyde isproduced by expressing a fatty aldehyde biosynthetic gene, for example,a carboxylic acid reductase gene (car gene), having a nucleotidesequence listed in FIGS. 6 and 8, as well as polynucleotide variantsthereof. In some instances, the fatty aldehyde biosynthetic gene encodesone or more of the amino acid motifs depicted in FIG. 7. For example,the gene can encode a polypeptide comprising SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, and SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13;SEQ ID NO:14; and/or SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, and SEQ IDNO:11. SEQ ID NO:7 includes a reductase domain; SEQ ID NO:8 and SEQ IDNO:14 include a NADP binding domain; SEQ ID NO:9 includes aphosphopantetheine attachment site; and SEQ ID NO:10 includes an AMPbinding domain.

In other instances, a fatty alcohol is produced by expressing a fattyalcohol biosynthetic gene, for example, having a nucleotide sequencelisted in FIG. 10, or a variant thereof.

Variants can be naturally occurring or created in vitro. In particular,such variants can be created using genetic engineering techniques, suchas site directed mutagenesis, random chemical mutagenesis, ExonucleaseIII deletion procedures, or standard cloning techniques. Alternatively,such variants, fragments, analogs, or derivatives can be created usingchemical synthesis or modification procedures.

Methods of making variants are well known in the art. These includeprocedures in which nucleic acid sequences obtained from naturalisolates are modified to generate nucleic acids that encode polypeptideshaving characteristics that enhance their value in industrial orlaboratory applications. In such procedures, a large number of variantsequences having one or more nucleotide differences with respect to thesequence obtained from the natural isolate are generated andcharacterized. Typically, these nucleotide differences result in aminoacid changes with respect to the polypeptides encoded by the nucleicacids from the natural isolates.

For example, variants can be created using error prone PCR (see, e.g.,Leung et al., Technique 1:11-15, 1989; and Caldwell et al., PCR MethodsApplic. 2:28-33, 1992). In error prone PCR, PCR is performed underconditions where the copying fidelity of the DNA polymerase is low, suchthat a high rate of point mutations is obtained along the entire lengthof the PCR product. Briefly, in such procedures, nucleic acids to bemutagenized (e.g., a fatty aldehyde biosynthetic polynucleotidesequence), are mixed with PCR primers, reaction buffer, MgCl₂, MnCl₂,Taq polymerase, and an appropriate concentration of dNTPs for achievinga high rate of point mutation along the entire length of the PCRproduct. For example, the reaction can be performed using 20 fmoles ofnucleic acid to be mutagenized (e.g., a fatty aldehyde biosyntheticpolynucleotide sequence), 30 pmole of each PCR primer, a reaction buffercomprising 50 mM KCl, 10 mM Tris HCl (pH 8.3), and 0.01% gelatin, 7 mMMgCl₂, 0.5 mM MnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mMdATP, 1 mM dCTP, and 1 mM dTTP. PCR can be performed for 30 cycles of94° C. for 1 min, 45° C. for 1 min, and 72° C. for 1 min. However, itwill be appreciated that these parameters can be varied as appropriate.The mutagenized nucleic acids are then cloned into an appropriate vectorand the activities of the polypeptides encoded by the mutagenizednucleic acids are evaluated.

Variants can also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described in, for example, Reidhaar-Olsonet al., Science 241:53-57, 1988. Briefly, in such procedures a pluralityof double stranded oligonucleotides bearing one or more mutations to beintroduced into the cloned DNA are synthesized and inserted into thecloned DNA to be mutagenized (e.g., a fatty aldehyde biosyntheticpolynucleotide sequence). Clones containing the mutagenized DNA arerecovered, and the activities of the polypeptides they encode areassessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, for example, U.S.Pat. No. 5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different, but highly related, DNA sequence invitro as a result of random fragmentation of the DNA molecule based onsequence homology. This is followed by fixation of the crossover byprimer extension in a PCR reaction. Sexual PCR mutagenesis is describedin, for example, Stemmer, PNAS, USA 91:10747-10751, 1994.

Variants can also be created by in vivo mutagenesis. In someembodiments, random mutations in a nucleic acid sequence are generatedby propagating the sequence in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type strain. Propagating a DNA sequence (e.g., a fattyaldehyde biosynthetic polynucleotide sequence) in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described in, forexample, PCT Publication No. WO 91/16427.

Variants can also be generated using cassette mutagenesis. In cassettemutagenesis, a small region of a double stranded DNA molecule isreplaced with a synthetic oligonucleotide “cassette” that differs fromthe native sequence. The oligonucleotide often contains a completelyand/or partially randomized native sequence.

Recursive ensemble mutagenesis can also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(i.e., protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described in, for example, Arkin et al., PNAS, USA89:7811-7815, 1992.

In some embodiments, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is describedin, for example, Delegrave et al., Biotech. Res. 11:1548-1552, 1993.Random and site-directed mutagenesis are described in, for example,Arnold, Curr. Opin. Biotech. 4:450-455, 1993.

In some embodiments, variants are created using shuffling procedureswherein portions of a plurality of nucleic acids that encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences that encode chimeric polypeptides as described in, forexample, U.S. Pat. Nos. 5,965,408 and 5,939,250.

Polynucleotide variants also include nucleic acid analogs. Nucleic acidanalogs can be modified at the base moiety, sugar moiety, or phosphatebackbone to improve, for example, stability, hybridization, orsolubility of the nucleic acid. Modifications at the base moiety includedeoxyuridine for deoxythymidine and 5-methyl-2′-deoxycytidine or5-bromo-2′-doxycytidine for deoxycytidine. Modifications of the sugarmoiety include modification of the 2′ hydroxyl of the ribose sugar toform 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphatebackbone can be modified to produce morpholino nucleic acids, in whicheach base moiety is linked to a six-membered, morpholino ring, orpeptide nucleic acids, in which the deoxyphosphate backbone is replacedby a pseudopeptide backbone and the four bases are retained. (See, e.g.,Summerton et al., Antisense Nucleic Acid Drug Dev. (1997) 7:187-195; andHyrup et al., Bioorgan. Med. Chem. (1996) 4:5-23.) In addition, thedeoxyphosphate backbone can be replaced with, for example, aphosphorothioate or phosphorodithioate backbone, a phosphoroamidite, oran alkyl phosphotriester backbone.

Any polynucleotide sequence encoding a homolog listed in FIGS. 6 and 8,or a variant thereof, can be used as a fatty aldehyde biosyntheticpolynucleotide in the methods described herein. Any polynucleotidesequence listed in FIG. 10, or a variant, can be used as a fatty alcoholbiosynthetic polynucleotide in the methods described herein.

Fatty Aldehyde Biosynthetic Polypeptides, Fatty Alcohol BiosyntheticPolypeptide, and Variants

The methods described herein can also be used to produce fatty alcohols,for example, from fatty aldehydes. In some instances, the fatty aldehydeis produced by a fatty aldehyde biosynthetic polypeptide having an aminoacid sequence listed in FIGS. 6 and 8, as well as polypeptide variantsthereof. In some instances, a fatty aldehyde biosynthetic polypeptide isone that includes one or more of the amino acid motifs depicted in FIG.7. For example, the polypeptide can include the amino acid sequences ofSEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. In othersituations, the polypeptide includes one or more of SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, and SEQ ID NO:14. In yet other instances, thepolypeptide includes the amino acid sequences of SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:10, and SEQ ID NO:11. SEQ ID NO:7 includes a reductasedomain; SEQ ID NO:8 and SEQ ID NO:14 include a NADP binding domain; SEQID NO:9 includes a phosphopantetheine attachment site; and SEQ ID NO:10includes an AMP binding domain.

In other instances, the methods described herein can be used to producefatty alcohols using a fatty alcohol biosynthetic polypeptide having anamino acid sequence listed in FIG. 10, as well as polypeptide variantsthereof.

Biosynthetic polypeptide variants can be variants in which one or moreamino acid residues are substituted with a conserved or non-conservedamino acid residue (preferably a conserved amino acid residue). Suchsubstituted amino acid residue may or may not be one encoded by thegenetic code.

Conservative substitutions are those that substitute a given amino acidin a polypeptide by another amino acid of similar characteristics.Typical conservative substitutions are the following replacements:replacement of an aliphatic amino acid, such as alanine, valine,leucine, and isoleucine, with another aliphatic amino acid; replacementof a serine with a threonine or vice versa; replacement of an acidicresidue, such as aspartic acid and glutamic acid, with another acidicresidue; replacement of a residue bearing an amide group, such asasparagine and glutamine, with another residue bearing an amide group;exchange of a basic residue, such as lysine and arginine, with anotherbasic residue; and replacement of an aromatic residue, such asphenylalanine and tyrosine, with another aromatic residue.

Other polypeptide variants are those in which one or more amino acidresidues include a substituent group. Still other polypeptide variantsare those in which the polypeptide is associated with another compound,such as a compound to increase the half-life of the polypeptide (e.g.,polyethylene glycol).

Additional polypeptide variants are those in which additional aminoacids are fused to the polypeptide, such as a leader sequence, asecretory sequence, a proprotein sequence, or a sequence whichfacilitates purification, enrichment, or stabilization of thepolypeptide.

In some instances, the polypeptide variants retain the same biologicalfunction as a polypeptide having an amino acid sequence listed in FIGS.6 and 8 (e.g., retain fatty aldehyde biosynthetic activity, such ascarboxylic acid or fatty acid reductase activity), or listed in FIG. 10(e.g., retain fatty alcohol biosynthetic activity, such as fatty alcoholdehydrogenase activity) and have amino acid sequences substantiallyidentical thereto.

In other instances, the polypeptide variants have at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or more than about 95% homology toan amino acid sequence listed in FIGS. 6, 8, and/or 10. In anotherembodiment, the polypeptide variants include a fragment comprising atleast about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof.

The polypeptide variants or fragments thereof can be obtained byisolating nucleic acids encoding them using techniques described hereinor by expressing synthetic nucleic acids encoding them. Alternatively,polypeptide variants or fragments thereof can be obtained throughbiochemical enrichment or purification procedures. The sequence ofpolypeptide variants or fragments can be determined by proteolyticdigestion, gel electrophoresis, and/or microsequencing. The sequence ofthe polypeptide variants or fragments can then be compared to an aminoacid sequence listed in FIGS. 6, 8, and/or 10 using any of the programsdescribed herein.

The polypeptide variants and fragments thereof can be assayed for fattyaldehyde-producing activity and/or fatty alcohol-producing activityusing routine methods. For example, the polypeptide variants or fragmentcan be contacted with a substrate (e.g., a fatty acid, a fatty acidderivative substrate, or other substrate described herein) underconditions that allow the polypeptide variant to function. A decrease inthe level of the substrate or an increase in the level of a fattyaldehyde can be measured to determine fatty aldehyde-producing activity.A decrease in the level of the substrate or an increase in the level ofa fatty alcohol can be measured to determine fatty alcohol-producingactivity.

Antibodies to Biosynthetic Polypeptides

The fatty aldehyde biosynthetic polypeptides described herein can alsobe used to produce antibodies directed against fatty aldehydebiosynthetic polypeptides. Such antibodies can be used, for example, todetect the expression of a fatty aldehyde biosynthetic polypeptide orfatty alcohol biosynthetic polypeptide using methods known in the art.The antibody can be, for example, a polyclonal antibody; a monoclonalantibody or antigen binding fragment thereof; a modified antibody suchas a chimeric antibody, reshaped antibody, humanized antibody, orfragment thereof (e.g., Fab′, Fab, F(ab′)₂); or a biosynthetic antibody,for example, a single chain antibody, single domain antibody (DAB), Fv,single chain Fv (scFv), or the like.

Methods of making and using polyclonal and monoclonal antibodies aredescribed, for example, in Harlow et al., Using Antibodies: A LaboratoryManual: Portable Protocol I. Cold Spring Harbor Laboratory (Dec. 1,1998). Methods for making modified antibodies and antibody fragments(e.g., chimeric antibodies, reshaped antibodies, humanized antibodies,or fragments thereof, e.g., Fab′, Fab, F(ab′)₂ fragments); orbiosynthetic antibodies (e.g., single chain antibodies, single domainantibodies (DABs), Fv, single chain Fv (scFv), and the like), are knownin the art and can be found, for example, in Zola, MonoclonalAntibodies: Preparation and Use of Monoclonal Antibodies and EngineeredAntibody Derivatives, Springer Verlag (Dec. 15, 2000; 1st edition).

Substrates

The compositions and methods described herein can be used to producefatty alcohols, for example, from fatty aldehydes, which themselves canbe produced from an appropriate substrate. While not wishing to be boundby theory, it is believed that the fatty aldehyde biosyntheticpolypeptides described herein produce fatty aldehydes from substratesvia a reduction mechanism. In some instances, the substrate is a fattyacid derivative (e.g., a fatty acid), and a fatty aldehyde havingparticular branching patterns and carbon chain length can be producedfrom a fatty acid derivative having those characteristics that wouldresult in a particular fatty aldehyde. Through an additional reactionmechanism, the fatty aldehyde can be converted into the desired fattyalcohol (e.g., by a fatty alcohol biosynthetic polypeptide describedherein).

Accordingly, each step within a biosynthetic pathway that leads to theproduction of a fatty acid derivative substrate can be modified toproduce or overproduce the substrate of interest. For example, knowngenes involved in the fatty acid biosynthetic pathway or the fattyaldehyde pathway can be expressed, overexpressed, or attenuated in hostcells to produce a desired substrate (see, e.g., PCT/US08/058,788).Exemplary genes are provided in FIG. 9.

Synthesis of Substrates

Fatty acid synthase (FAS) is a group of polypeptides that catalyze theinitiation and elongation of acyl chains (Marrakchi et al., BiochemicalSociety, 30:1050-1055, 2002). The acyl carrier protein (ACP) along withthe enzymes in the FAS pathway control the length, degree of saturation,and branching of the fatty acid derivatives produced. The fatty acidbiosynthetic pathway involves the precursors acetyl-CoA and malonyl-CoA.The steps in this pathway are catalyzed by enzymes of the fatty acidbiosynthesis (fab) and acetyl-CoA carboxylase (acc) gene families (see,e.g., Heath et al., Prog. Lipid Res. 40(6):467-97 (2001)).

Host cells can be engineered to express fatty acid derivative substratesby recombinantly expressing or overexpressing one or more fatty acidsynthase genes, such as acetyl-CoA and/or malonyl-CoA synthase genes.For example, to increase acetyl-CoA production, one or more of thefollowing genes can be expressed in a host cell: pdh (a multienzymecomplex comprising aceEF (which encodes the E1p dehydrogenase component,the E2p dihydrolipoamide acyltransferase component of the pyruvate and2-oxoglutarate dehydrogenase complexes, and lpc), panK, fabH, fabB,fabD, fabG, acpP, and fabF. Exemplary GenBank accession numbers forthese genes are: pdh (BAB34380, AAC73227, AAC73226), panK (also known asCoA, AAC76952), aceEF (AAC73227, AAC73226), fabH (AAC74175), fabB(P0A953), fabD (AAC74176), fabG (AAC74177), acpP (AAC74178), fabF(AAC74179). Additionally, the expression levels of fadE, gpsA, ldhA,pflb, adhE, pta, poxB, ackA, and/or ackB can be attenuated orknocked-out in an engineered host cell by transformation withconditionally replicative or non-replicative plasmids containing null ordeletion mutations of the corresponding genes or by substitutingpromoter or enhancer sequences. Exemplary GenBank accession numbers forthese genes are: fadE (AAC73325), gspA (AAC76632), ldhA (AAC74462), pflb(AAC73989), adhE (AAC74323), pta (AAC75357), poxB (AAC73958), ackA(AAC75356), and ackB (BAB81430). The resulting host cells will haveincreased acetyl-CoA production levels when grown in an appropriateenvironment.

Malonyl-CoA overexpression can be affected by introducing accABCD (e.g.,accession number AAC73296, EC 6.4.1.2) into a host cell. Fatty acids canbe further overexpressed in host cells by introducing into the host cella DNA sequence encoding a lipase (e.g., accession numbers CAA89087,CAA98876).

In addition, inhibiting PlsB can lead to an increase in the levels oflong chain acyl-ACP, which will inhibit early steps in the pathway(e.g., accABCD, fabH, and fabI). The plsB (e.g., accession numberAAC77011) D311E mutation can be used to increase the amount of availablefatty acids.

In addition, a host cell can be engineered to overexpress a sfa gene(suppressor of fabA, e.g., accession number AAN79592) to increaseproduction of monounsaturated fatty acids (Rock et al., J. Bacteriology178:5382-5387, 1996).

The chain length of a fatty acid derivative substrate can be selectedfor by modifying the expression of selected thioesterases. Thioesteraseinfluences the chain length of fatty acids produced. Hence, host cellscan be engineered to express, overexpress, have attenuated expression,or not to express one or more selected thioesterases to increase theproduction of a preferred fatty acid derivative substrate. For example,C₁₀ fatty acids can be produced by expressing a thioesterase that has apreference for producing C₁₀ fatty acids and attenuating thioesterasesthat have a preference for producing fatty acids other than C₁₀ fattyacids (e.g., a thioesterase which prefers to produce C₁₄ fatty acids).This would result in a relatively homogeneous population of fatty acidsthat have a carbon chain length of 10. In other instances, C₁₄ fattyacids can be produced by attenuating endogenous thioesterases thatproduce non-C₁₄ fatty acids and expressing the thioesterases that useC₁₄-ACP. In some situations, C₁₂ fatty acids can be produced byexpressing thioesterases that use C₁₂-ACP and attenuating thioesterasesthat produce non-C₁₂ fatty acids. Acetyl-CoA, malonyl-CoA, and fattyacid overproduction can be verified using methods known in the art, forexample, by using radioactive precursors, HPLC, or GC-MS subsequent tocell lysis. Non-limiting examples of thioesterases that can be used inthe methods described herein are listed in Table 1.

TABLE 1 Thioesterases Accession Number Source Organism Gene AAC73596 E.coli tesA without leader sequence AAC73555 E. coli tesB Q41635, AAA34215Umbellularia california fatB AAC49269 Cuphea hookeriana fatB2 Q39513;AAC72881 Cuphea hookeriana fatB3 Q39473, AAC49151 Cinnamonum camphorumfatB CAA85388 Arabidopsis thaliana fatB [M141T]* NP 189147; NP 193041Arabidopsis thaliana fatA CAC39106 Bradyrhiizobium japonicum fatAAAC72883 Cuphea hookeriana fatA AAL79361 Helianthus annus fatA1 *Mayeret al., BMC Plant Biology 7: 1-11, 2007

In other instances, a fatty aldehyde biosynthetic polypeptide, variant,or a fragment thereof, is expressed in a host cell that contains anaturally occurring mutation that results in an increased level of fattyacids in the host cell. In some instances, the host cell is geneticallyengineered to increase the level of fatty acids in the host cellrelative to a corresponding wild-type host cell. For example, the hostcell can be genetically engineered to express a reduced level of anacyl-CoA synthase relative to a corresponding wild-type host cell. Inone embodiment, the level of expression of one or more genes (e.g., anacyl-CoA synthase gene) is reduced by genetically engineering a “knockout” host cell.

Any known acyl-CoA synthase gene can be reduced or knocked out in a hostcell. Non-limiting examples of acyl-CoA synthase genes include fadD,fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.Specific examples of acyl-CoA synthase genes include fadDD35 from M.tuberculosis H37Rv [NP_(—)217021], fadDD22 from M. tuberculosis H37Rv[NP_(—)217464], fadD from E. coli [NP_(—)416319], fadK from E. coli[YP_(—)416216], fadD from Acinetobacter sp. ADP1 [YP_(—)045024], fadDfrom Haemophilus influenza RdkW20 [NP_(—)438551], fadD fromRhodopseudomonas palustris Bis B18 [YP_(—)533919], BH3101 from Bacillushalodurans C-125 [NP_(—)243969], Pfl-4354 from Pseudomonas fluorescensPfo-1 [YP_(—)350082], EAV15023 from Comamonas testosterone KF-1[ZP_(—)01520072], yhfL from B. subtilis [NP_(—)388908], fadD1 from P.aeruginosa PAO1 [NP_(—)251989], fadD1 from Ralstonia solanacearum GM11000 [NP_(—)520978], fadD2 from P. aeruginosa PAO1 [NP_(—)251990], thegene encoding the protein ZP_(—)01644857 from Stenotrophomonasmaltophilia R551-3, faa3p from Saccharomyces cerevisiae [NP_(—)012257],faalp from Saccharomyces cerevisiae [NP_(—)014962], lcfA from Bacillussubtilis [CAA99571], or those described in Shockey et al., Plant.Physiol. 129:1710-1722, 2002; Caviglia et al., J. Biol. Chem.279:1163-1169, 2004; Knoll et al., J. Biol. Chem. 269(23):16348-56,1994; Johnson et al., J. Biol. Chem. 269: 18037-18046, 1994; and Blacket al., J. Biol Chem. 267: 25513-25520, 1992.

Formation of Branched Fatty Alcohols

Fatty alcohols can be produced from fatty aldehydes that contain branchpoints by using branched fatty acid derivatives as substrates for afatty aldehyde biosynthetic polypeptide described herein. For example,although E. coli naturally produces straight chain fatty acids (sFAs),E. coli can be engineered to produce branched chain fatty acids (brFAs)by introducing and expressing or overexpressing genes that providebranched precursors in the E. coli (e.g., bkd, ilv, icm, and fab genefamilies). Additionally, a host cell can be engineered to express oroverexpress genes encoding proteins for the elongation of brFAs (e.g.,ACP, FabF, etc.) and/or to delete or attenuate the corresponding hostcell genes that normally lead to sFAs.

The first step in forming brFAs is the production of the correspondingα-keto acids by a branched-chain amino acid aminotransferase. Host cellsmay endogenously include genes encoding such enzymes or such genes canbe recombinantly introduced. E. coli, for example, endogenouslyexpresses such an enzyme, IlvE (EC 2.6.1.42; GenBank accessionYP_(—)026247). In some host cells, a heterologous branched-chain aminoacid aminotransferase may not be expressed. However, E. coli IlvE or anyother branched-chain amino acid aminotransferase (e.g., IlvE fromLactococcus lactis (GenBank accession AAF34406), IlvE from Pseudomonasputida (GenBank accession NP_(—)745648), or IlvE from Streptomycescoelicolor (GenBank accession NP_(—)629657)), if not endogenous, can beintroduced.

In another embodiment, the production of α-keto acids can be achieved byusing the methods described in Atsumi et al., Nature 451:86-89, 2008.For example, 2-ketoisovalerate can be produced by overexpressing thegenes encoding IlvI, IlvH, IlvC, or IlvD. In another example,2-keto-3-methyl-valerate can be produced by overexpressing the genesencoding IlvA and IlvI, IlvH (or AlsS of Bacillus subtilis), IlvC, IlvD,or their corresponding homologs. In a further embodiment,2-keto-4-methyl-pentanoate can be produced by overexpressing the genesencoding IlvI, IlvH, IlvC, IlvD and LeuA, LeuB, LeuC, LeuD, or theircorresponding homologs.

The second step is the oxidative decarboxylation of the α-keto acids tothe corresponding branched-chain acyl-CoA. This reaction can becatalyzed by a branched-chain α-keto acid dehydrogenase complex (bkd; EC1.2.4.4.) (Denoya et al., J. Bacteriol. 177:3504, 1995), which consistsof E1α/β (decarboxylase), E2 (dihydrolipoyl transacylase), and E3(dihydrolipoyl dehydrogenase) subunits. These branched-chain α-keto aciddehydrogenase complexes are similar to pyruvate and α-ketoglutaratedehydrogenase complexes. Any microorganism that possesses brFAs and/orgrows on branched-chain amino acids can be used as a source to isolatebkd genes for expression in host cells, for example, E. coli.Furthermore, E. coli has the E3 component as part of its pyruvatedehydrogenase complex (lpd, EC 1.8.1.4, GenBank accession NP_(—)414658).Thus, it may be sufficient to express only the E1 α/β and E2 bkd genes.Table 2 lists non-limiting examples of bkd genes from severalmicroorganisms that can be recombinantly introduced and expressed in ahost cell to provide branched-chain acyl-CoA precursors.

TABLE 2 Bkd genes from selected microorganisms GenBank Organism GeneAccession # Streptomyces coelicolor bkdA1 (E1α) NP_628006 bkdB1 (E1β)NP_628005 bkdC1 (E2) NP_638004 Streptomyces coelicolor bkdA2 (E1α)NP_733618 bkdB2 (E1β) NP_628019 bkdC2 (E2) NP_628018 Streptomycesavermitilis bkdA (E1a) BAC72074 bkdB (E1b) BAC72075 bkdC (E2) BAC72076Streptomyces avermitilis bkdF (E1α) BAC72088 bkdG (E1β) BAC72089 bkdH(E2) BAC72090 Bacillus subtilis bkdAA (E1α) NP_390288 bkdAB (E1β)NP_390288 bkdB (E2) NP_390288 Pseudomonas putida bkdA1 (E1α) AAA65614bkdA2 (E1β) AAA65615 bkdC (E2) AAA65617

In another example, isobutyryl-CoA can be made in a host cell, forexample in E. coli, through the coexpression of a crotonyl-CoA reductase(Ccr, EC 1.6.5.5, 1.1.1.1) and isobutyryl-CoA mutase (large subunitIcmA, EC 5.4.99.2; small subunit IcmB, EC 5.4.99.2) (Han and Reynolds,J. Bacteriol. 179:5157, 1997). Crotonyl-CoA is an intermediate in fattyacid biosynthesis in E. coli and other microorganisms. Non-limitingexamples of ccr and icm genes from selected microorganisms are listed inTable 3.

TABLE 3 Ccr and icm genes from selected microorganisms GenBank OrganismGene Accession # Streptomyces coelicolor ccr NP_630556 icmA NP_629554icmB NP_630904 Streptomyces cinnamonensis ccr AAD53915 icmA AAC08713icmB AJ246005

In addition to expression of the bkd genes, the initiation of brFAbiosynthesis utilizes β-ketoacyl-acyl-carrier-protein synthase III(FabH, EC 2.3.1.41) with specificity for branched chain acyl-CoAs (Li etal., J. Bacteriol. 187:3795-3799, 2005). Non-limiting examples of suchFabH enzymes are listed in Table 4. fabH genes that are involved infatty acid biosynthesis of any brFA-containing microorganism can beexpressed in a host cell. The Bkd and FabH enzymes from host cells thatdo not naturally make brFA may not support brFA production. Therefore,bkd and fabH can be expressed recombinantly. Vectors containing the bkdand fabH genes can be inserted into such a host cell. Similarly, theendogenous level of Bkd and FabH production may not be sufficient toproduce brFA. In this case, they can be overexpressed. Additionally,other components of the fatty acid biosynthesis pathway can be expressedor overexpressed, such as acyl carrier proteins (ACPs) andβ-ketoacyl-acyl-carrier-protein synthase II (fabF, EC 2.3.1.41)(non-limiting examples of candidates are listed in Table 4). In additionto expressing these genes, some genes in the endogenous fatty acidbiosynthesis pathway can be attenuated in the host cell (e.g., the E.coli genes fabH (GenBank accession # NP_(—)415609) and/or fabF (GenBankaccession # NP_(—)415613)).

TABLE 4 FabH, ACP and fabF genes from selected microorganisms with brFAsGenBank Organism Gene Accession # Streptomyces coelicolor fabH1NP_626634 acp NP_626635 fabF NP_626636 Streptomyces avermitilis fabH3NP_823466 fabC3 (acp) NP_823467 fabF NP_823468 Bacillus subtilis fabH_ANP_389015 fabH_B NP_388898 acp NP_389474 fabF NP_389016 StenotrophomonasSmalDRAFT_0818 (fabH) ZP_01643059 maltophilia SmalDRAFT_0821 (acp)ZP_01643063 SmalDRAFT_0822 (fabF) ZP_01643064 Legionella pneumophilafabH YP_123672 acp YP_123675 fabF YP_123676

Formation of Cyclic Fatty Alcohols

Cyclic fatty alcohols can be produced from cyclic fatty aldehydes usingcyclic fatty acid derivatives as substrates for a fatty aldehydebiosynthetic polypeptide described herein. To produce cyclic fatty acidderivative substrates, genes that provide cyclic precursors (e.g., theans, chc, and plm gene families) can be introduced into the host celland expressed to allow initiation of fatty acid biosynthesis from cyclicprecursors. For example, to convert a host cell, such as E. coli, intoone capable of synthesizing co-cyclic fatty acids (cyFA), a gene thatprovides the cyclic precursor cyclohexylcarbonyl-CoA (CHC-CoA) (Cropp etal., Nature Biotech. 18:980-983, 2000) can be introduced and expressedin the host cell. Non-limiting examples of genes that provide CHC-CoA inE. coli include: ansJ, ansK, ansL, chcA, and ansM from the ansatrieningene cluster of Streptomyces collinus (Chen et al., Eur. J. Biochem.261: 98-107, 1999) or plmJ, plmK, plmL, chcA, and plmM from thephoslactomycin B gene cluster of Streptomyces sp. HK803 (Palaniappan etal., J. Biol. Chem. 278:35552-35557, 2003) together with the chcB gene(Patton et al., Biochem. 39:7595-7604, 2000) from S. collinus, S.avermitilis, or S. coelicolor (see Table 5). The genes listed in Table 4can then be expressed to allow initiation and elongation of ω-cyclicfatty acids. Alternatively, the homologous genes can be isolated frommicroorganisms that make cyFA and expressed in a host cell (e.g., E.coli).

TABLE 5 Genes for the synthesis of CHC-CoA GenBank Organism GeneAccession # Streptomyces collinus ansJK U72144* ansL chcA ansM chcBAF268489 Streptomyces sp. HK803 pmlJK AAQ84158 pmlL AAQ84159 chcAAAQ84160 pmlM AAQ84161 Streptomyces coelicolor chcB/caiD NP_629292Streptomyces avermitilis chcB/caiD NP_629292 *Only chcA is annotated inGenBank entry U72144, ansJKLM are according to Chen et al. (Eur. J.Biochem. 261: 98-107, 1999).

The genes listed in Table 4 (fabH, acp, and fabF) allow initiation andelongation of w-cyclic fatty acids because they have broad substratespecificity. If the coexpression of any of these genes with the geneslisted in Table 5 does not yield cyFA, then fabH, acp, and/or fabFhomologs from microorganisms that make cyFAs (e.g., those listed inTable 6) can be isolated (e.g., by using degenerate PCR primers orheterologous DNA sequence probes) and coexpressed.

TABLE 6 Non-limiting examples of microorganisms that contain ω-cyclicfatty acids Organism Reference Curtobacterium pusillum ATCC19096Alicyclobacillus acidoterrestris ATCC49025 Alicyclobacillusacidocaldarius ATCC27009 Alicyclobacillus cycloheptanicus * Moore, J.Org. Chem. 62: pp. 2173, 1997 * Uses cycloheptylcarbonyl-CoA and notcyclohexylcarbonyl-CoA as precursor for cyFA biosynthesis.

Fatty Alcohol Saturation Levels

The degree of saturation in fatty acids (which can then be convertedinto fatty aldehydes and then fatty alcohols as described herein) can becontrolled by regulating the degree of saturation of fatty acidintermediates. For example, the sfa, gns, and fab families of genes canbe expressed, overexpressed, or expressed at reduced levels, to controlthe saturation of fatty acids. FIG. 9 lists non-limiting examples ofgenes in these gene families that may be used in the methods and hostcells described herein.

For example, host cells can be engineered to produce unsaturated fattyacids by engineering the production host to overexpress fabB or bygrowing the production host at low temperatures (e.g., less than 37°C.). FabB has preference to cis-δ3decenoyl-ACP and results inunsaturated fatty acid production in E. coli. Overexpression of fabBresults in the production of a significant percentage of unsaturatedfatty acids (de Mendoza et al., J. Biol. Chem. 258:2098-2101, 1983). Thegene fabB may be inserted into and expressed in host cells not naturallyhaving the gene. These unsaturated fatty acids can then be used asintermediates in host cells that are engineered to produce fatty acidderivatives, such as fatty aldehydes.

In other instances, a repressor of fatty acid biosynthesis, for example,fabR (GenBank accession NP_(—)418398), can be deleted, which will alsoresult in increased unsaturated fatty acid production in E. coli (Zhanget al., J. Biol. Chem. 277:15558, 2002). Similar deletions may be madein other host cells. A further increase in unsaturated fatty acids maybe achieved, for example, by overexpressing fabM (trans-2,cis-3-decenoyl-ACP isomerase, GenBank accession DAA05501) and controlledexpression of fabK (trans-2-enoyl-ACP reductase II, GenBank accessionNP_(—)357969) from Streptococcus pneumoniae (Marrakchi et al., J. Biol.Chem. 277: 44809, 2002), while deleting E. coli fabI (trans-2-enoyl-ACPreductase, GenBank accession NP_(—)415804). In some examples, theendogenous fabF gene can be attenuated, thus increasing the percentageof palmitoleate (C16:1) produced.

In yet other examples, host cells can be engineered to produce saturatedfatty acids by reducing the expression of an sfa, gns, and/or fab gene.

In some instances, a host cell can be engineered to express anattenuated level of a dehydratase/isomerase and/or a ketoacyl-ACPsynthase. For example, a host cell can be engineered to express adecreased level of fabA and/or fabB. In some instances, the host cellcan be grown in the presence of unsaturated fatty acids. In otherinstances, the host cell can be further engineered to express oroverexpress a gene encoding a desaturase enzyme. One nonlimiting exampleof a desaturase is B. subtilis DesA (AF037430). Other genes encodingdesaturase enzymes are known in the art and can be used in the hostcells and methods described herein, such as desaturases that useacyl-ACP, such as hexadecanoyl-ACP or octadecanoyl-ACP. The saturatedfatty acids can be used to produce fatty acid derivatives, such as fattyaldehydes, and subsequently saturated fatty alcohols, as describedherein.

Production of Fatty Alcohols

A fatty aldehyde described herein can be converted into a fatty alcoholby an alcohol dehydrogenase. In some examples, a gene encoding a fattyaldehyde biosynthetic polypeptide described herein can be expressed in ahost cell that expresses an endogenous alcohol dehydrogenase capable ofconverting a fatty aldehyde produced by the fatty aldehyde biosyntheticpolypeptide into a corresponding fatty alcohol. In other instances, agene encoding a fatty alcohol biosynthetic polypeptide described herein,such as an amino acid sequence listed in FIG. 10 or a variant thereof,can be expressed in a host cell. Exemplary fatty alcohol biosyntheticgenes include, but are not limited to, AlrA of Acenitobacter sp. M-1 orAlrA homologs; and endogenous E. coli alcohol dehydrogenases such asDkgA (NP_(—)417-485), DkgB (NP_(—)414743), YjgB, (AAC77226), YdjL(AAC74846), YdjJ (NP_(—)416288), AdhP (NP_(—)415995), YhdH(NP_(—)417719), YahK (NP_(—)414859), YphC (AAC75598), and YqhD (Q46856).In other instances, a gene encoding a fatty alcohol biosyntheticpolypeptide can be co-expressed in a host cell with a gene encoding afatty aldehyde biosynthetic polypeptide described herein.

Genetic Engineering of Host Cells to Produce Fatty Alcohols

Various host cells can be used to produce fatty alcohols, as describedherein. A host cell can be any prokaryotic or eukaryotic cell. Forexample, a gene encoding a polypeptide described herein (e.g., a fattyaldehyde biosynthetic polypeptide and/or a fatty alcohol biosyntheticpolypeptide) can be expressed in bacterial cells (such as E. coli),insect cells, yeast, or mammalian cells (such as Chinese hamster ovarycells (CHO) cells, COS cells, VERO cells, BHK cells, HeLa cells, Cv1cells, MDCK cells, 293 cells, 3T3 cells, or PC12 cells). Other exemplaryhost cells include cells from the members of the genus Escherichia,Bacillus, Lactobacillus, Rhodococcus, Pseudomonas, Aspergillus,Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces,Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus,Trametes, Chrysosporium, Saccharomyces, Schizosaccharomyces, Yarrowia,or Streptomyces. Yet other exemplary host cells can be a Bacillus lentuscell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, aBacillus licheniformis cell, a Bacillus alkalophilus cell, a Bacilluscoagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, aBacillus thuringiensis cell, a Bacillus clausii cell, a Bacillusmegaterium cell, a Bacillus subtilis cell, a Bacillus amyloliquefacienscell, a Trichoderma koningii cell, a Trichoderma viride cell, aTrichoderma reesei cell, a Trichoderma longibrachiatum cell, anAspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillusfoetidus cell, an Aspergillus nidulans cell, an Aspergillus niger cell,an Aspergillus oryzae cell, a Humicola insolens cell, a Humicolalanuginose cell, a Rhizomucor miehei cell, a Mucor michei cell, aStreptomyces lividans cell, a Streptomyces murinus cell, or anActinomycetes cell. Other host cells are cyanobacterial host cells.

In a preferred embodiment, the host cell is an E. coli cell, aSaccharomyces cerevisiae cell, or a Bacillus subtilis cell. In a morepreferred embodiment, the host cell is from E. coli strains B, C, K, orW. Other suitable host cells are known to those skilled in the art.

Additional host cells that can be used in the methods described hereinare described in WO2009/111513 and WO2009/111672.

Various methods well known in the art can be used to geneticallyengineer host cells to produce fatty alcohols. The methods can includethe use of vectors, preferably expression vectors, containing a nucleicacid encoding a fatty aldehyde biosynthetic polypeptide and/or a fattyalcohol biosynthetic polypeptide described herein, polypeptide variant,or a fragment thereof. Those skilled in the art will appreciate avariety of viral vectors (for example, retroviral vectors, lentiviralvectors, adenoviral vectors, and adeno-associated viral vectors) andnon-viral vectors can be used in the methods described herein.

The recombinant expression vectors described herein include a nucleicacid described herein in a form suitable for expression of the nucleicacid in a host cell. The recombinant expression vectors can include oneor more control sequences, selected on the basis of the host cell to beused for expression. The control sequence is operably linked to thenucleic acid sequence to be expressed. Such control sequences aredescribed, for example, in Goeddel, Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Controlsequences include those that direct constitutive expression of anucleotide sequence in many types of host cells and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectorsdescribed herein can be introduced into host cells to producepolypeptides, including fusion polypeptides, encoded by the nucleicacids as described herein.

Recombinant expression vectors can be designed for expression of a geneencoding a fatty aldehyde biosynthetic polypeptide (or variant) and/or agene encoding a fatty alcohol biosynthetic polypeptide in prokaryotic oreukaryotic cells (e.g., bacterial cells, such as E. coli, insect cells(e.g., using baculovirus expression vectors), yeast cells, or mammaliancells). Suitable host cells are discussed further in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Alternatively, the recombinant expression vectorcan be transcribed and translated in vitro, for example, by using T7promoter regulatory sequences and T7 polymerase.

Expression of genes encoding polypeptides in prokaryotes, for example,E. coli, is most often carried out with vectors containing constitutiveor inducible promoters directing the expression of either fusion ornon-fusion polypeptides. Fusion vectors add a number of amino acids to apolypeptide encoded therein, usually to the amino terminus of therecombinant polypeptide. Such fusion vectors typically serve threepurposes: (1) to increase expression of the recombinant polypeptide; (2)to increase the solubility of the recombinant polypeptide; and (3) toaid in the purification of the recombinant polypeptide by acting as aligand in affinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant polypeptide. This enables separation of therecombinant polypeptide from the fusion moiety after purification of thefusion polypeptide. Examples of such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin, and enterokinase.Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith et al., Gene (1988) 67:31-40), pMAL (New England Biolabs, Beverly,Mass.), and pRITS (Pharmacia, Piscataway, N.J.), which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant polypeptide.

Examples of inducible, non-fusion E. coli expression vectors includepTrc (Amann et al., Gene (1988) 69:301-315) and pET 11d (Studier et al.,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990) 60-89). Target gene expression from the pTrcvector relies on host RNA polymerase transcription from a hybrid trp-lacfusion promoter. Target gene expression from the pET 11d vector relieson transcription from a T7 gn 10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant polypeptide expression is toexpress the polypeptide in a host cell with an impaired capacity toproteolytically cleave the recombinant polypeptide (see Gottesman, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin the host cell (Wada et al., Nucleic Acids Res. (1992) 20:2111-2118).Such alteration of nucleic acid sequences can be carried out by standardDNA synthesis techniques.

In another embodiment, the host cell is a yeast cell. In thisembodiment, the expression vector is a yeast expression vector. Examplesof vectors for expression in yeast S. cerevisiae include pYepSec1(Baldari et al., EMBO J. (1987) 6:229-234), pMFa (Kurjan et al., Cell(1982) 30:933-943), pJRY88 (Schultz et al., Gene (1987) 54:113-123),pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InvitrogenCorp, San Diego, Calif.).

Alternatively, a polypeptide described herein can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include, for example, the pAc series (Smith et al., Mol. CellBiol. (1983) 3:2156-2165) and the pVL series (Lucklow et al., Virology(1989) 170:31-39).

In yet another embodiment, the nucleic acids described herein can beexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, Nature(1987) 329:840) and pMT2PC (Kaufman et al., EMBO J. (1987) 6:187-195).When used in mammalian cells, the expression vector's control functionscan be provided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus, andSimian Virus 40. Other suitable expression systems for both prokaryoticand eukaryotic cells are described in chapters 16 and 17 of Sambrook etal., eds., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989.

Vectors can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” refer to a variety ofart-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in, for example, Sambrook et al. (supra).

For stable transformation of bacterial cells, it is known that,depending upon the expression vector and transformation technique used,only a small fraction of cells will take-up and replicate the expressionvector. In order to identify and select these transformants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) can beintroduced into the host cells along with the gene of interest.Selectable markers include those that confer resistance to drugs, suchas ampicillin, kanamycin, chloramphenicol, or tetracycline. Nucleicacids encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding a polypeptide described herein or canbe introduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) can be introducedinto the host cells along with the gene of interest. Preferredselectable markers include those which confer resistance to drugs, suchas G418, hygromycin, and methotrexate. Nucleic acids encoding aselectable marker can be introduced into a host cell on the same vectoras that encoding a polypeptide described herein or can be introduced ona separate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

Transport Proteins

Transport proteins can export polypeptides and organic compounds (e.g.,fatty alcohols) out of a host cell. Many transport and efflux proteinsserve to excrete a wide variety of compounds and can be naturallymodified to be selective for particular types of hydrocarbons.

Non-limiting examples of suitable transport proteins are ATP-BindingCassette (ABC) transport proteins, efflux proteins, and fatty acidtransporter proteins (FATP). Additional non-limiting examples ofsuitable transport proteins include the ABC transport proteins fromorganisms such as Caenorhabditis elegans, Arabidopsis thalania,Alkaligenes eutrophus, and Rhodococcus erythropolis. Exemplary ABCtransport proteins that can be used are listed in FIG. 9 (e.g., CER5,AtMRP5, AmiS2, and AtPGP1). Host cells can also be chosen for theirendogenous ability to secrete organic compounds. The efficiency oforganic compound production and secretion into the host cell environment(e.g., culture medium, fermentation broth) can be expressed as a ratioof intracellular product to extracellular product. In some examples, theratio can be about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5.

Fermentation

The production and isolation of fatty alcohols can be enhanced byemploying beneficial fermentation techniques. One method for maximizingproduction while reducing costs is increasing the percentage of thecarbon source that is converted to hydrocarbon products.

During normal cellular lifecycles, carbon is used in cellular functions,such as producing lipids, saccharides, proteins, organic acids, andnucleic acids. Reducing the amount of carbon necessary forgrowth-related activities can increase the efficiency of carbon sourceconversion to product. This can be achieved by, for example, firstgrowing host cells to a desired density (for example, a density achievedat the peak of the log phase of growth). At such a point, replicationcheckpoint genes can be harnessed to stop the growth of cells.Specifically, quorum sensing mechanisms (reviewed in Camilli et al.,Science 311:1113, 2006; Venturi FEMS Microbio. Rev. 30:274-291, 2006;and Reading et al., FEMS Microbiol. Lett. 254:1-11, 2006) can be used toactivate checkpoint genes, such as p53, p21, or other checkpoint genes.

Genes that can be activated to stop cell replication and growth in E.coli include umuDC genes. The overexpression of umuDC genes stops theprogression from stationary phase to exponential growth (Murli et al.,J. of Bact. 182:1127, 2000). UmuC is a DNA polymerase that can carry outtranslesion synthesis over non-coding lesions—the mechanistic basis ofmost UV and chemical mutagenesis. The umuDC gene products are involvedin the process of translesion synthesis and also serve as a DNA sequencedamage checkpoint. The umuDC gene products include UmuC, UmuD, umuD′,UmuD′₂C, UmuD′₂, and UmuD₂. Simultaneously, product-producing genes canbe activated, thus minimizing the need for replication and maintenancepathways to be used while a fatty aldehyde is being made. Host cells canalso be engineered to express umuC and umuD from E. coli in pBAD24 underthe prpBCDE promoter system through de novo synthesis of this gene withthe appropriate end-product production genes.

The percentage of input carbons converted to fatty alcohols can be acost driver. The more efficient the process is (i.e., the higher thepercentage of input carbons converted to fatty alcohols), the lessexpensive the process will be. For oxygen-containing carbon sources(e.g., glucose and other carbohydrate based sources), the oxygen must bereleased in the form of carbon dioxide. For every 2 oxygen atomsreleased, a carbon atom is also released leading to a maximaltheoretical metabolic efficiency of approximately 34% (w/w) (for fattyacid derived products). This figure, however, changes for other organiccompounds and carbon sources. Typical efficiencies in the literature areapproximately less than 5%. Host cells engineered to produce fattyalcohols can have greater than about 1, 3, 5, 10, 15, 20, 25, and 30%efficiency. In one example, host cells can exhibit an efficiency ofabout 10% to about 25%. In other examples, such host cells can exhibitan efficiency of about 25% to about 30%. In other examples, host cellscan exhibit greater than 30% efficiency.

The host cell can be additionally engineered to express recombinantcellulosomes, such as those described in PCT application numberPCT/US2007/003736. These cellulosomes can allow the host cell to usecellulosic material as a carbon source. For example, the host cell canbe additionally engineered to express invertases (EC 3.2.1.26) so thatsucrose can be used as a carbon source. Similarly, the host cell can beengineered using the teachings described in U.S. Pat. Nos. 5,000,000;5,028,539; 5,424,202; 5,482,846; and 5,602,030; so that the host cellcan assimilate carbon efficiently and use cellulosic materials as carbonsources.

In one example, the fermentation chamber can enclose a fermentation thatis undergoing a continuous reduction. In this instance, a stablereductive environment can be created. The electron balance can bemaintained by the release of carbon dioxide (in gaseous form). Effortsto augment the NAD/H and NADP/H balance can also facilitate instabilizing the electron balance. The availability of intracellularNADPH can also be enhanced by engineering the host cell to express anNADH:NADPH transhydrogenase. The expression of one or more NADH:NADPHtranshydrogenases converts the NADH produced in glycolysis to NADPH,which can enhance the production of fatty alcohols.

For small scale production, the engineered host cells can be grown inbatches of, for example, about 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L;fermented; and induced to express desired fatty aldehyde biosyntheticgenes and/or an alcohol dehydrogenase genes based on the specific genesencoded in the appropriate plasmids. For large scale production, theengineered host cells can be grown in batches of about 10 L, 100 L, 1000L, 10,000 L, 100,000 L, 1,000,000 L or larger; fermented; and induced toexpress desired fatty aldehyde biosynthetic genes and/or alcoholdehydrogenase genes based on the specific genes encoded in theappropriate plasmids or incorporated into the host cell's genome.

For example, a suitable production host, such as E. coli cells,harboring plasmids containing the desired genes or having the genesintegrated in its chromosome can be incubated in a suitable reactor, forexample a 1 L reactor, for 20 hours at 37° C. in M9 medium supplementedwith 2% glucose, carbenicillin, and chloramphenicol. When the OD₆₀₀ ofthe culture reaches 0.9, the production host can be induced with IPTGalcohol After incubation, the spent media can be extracted and theorganic phase can be examined for the presence of fatty alcohols usingGC-MS.

In some instances, after the first hour of induction, aliquots of nomore than about 10% of the total cell volume can be removed each hourand allowed to sit without agitation to allow the fatty alcohols to riseto the surface and undergo a spontaneous phase separation orprecipitation. The fatty alcohol component can then be collected, andthe aqueous phase returned to the reaction chamber. The reaction chambercan be operated continuously. When the OD₆₀₀ drops below 0.6, the cellscan be replaced with a new batch grown from a seed culture.

Producing Fatty Alcohols Using Cell-Free Methods

In some methods described herein, a fatty alcohol can be produced usinga purified polypeptide (e.g., a fatty alcohol biosynthetic polypeptide)described herein and a substrate (e.g., fatty aldehyde), produced, forexample, by a method described herein. For example, a host cell can beengineered to express a fatty alcohol biosynthetic polypeptide orvariant as described herein. The host cell can be cultured underconditions suitable to allow expression of the polypeptide. Cell freeextracts can then be generated using known methods. For example, thehost cells can be lysed using detergents or by sonication. The expressedpolypeptides can be purified using known methods. After obtaining thecell free extracts, substrates described herein can be added to the cellfree extracts and maintained under conditions to allow conversion of thesubstrates (e.g., fatty aldehydes) to fatty alcohols. The fatty alcoholscan then be separated and purified using known techniques.

In some instances, a fatty aldehyde described herein can be convertedinto a fatty alcohol by contacting the fatty aldehyde with a fattyalcohol biosynthetic polypeptide listed in FIG. 10, or a variantthereof.

Post-Production Processing

The fatty alcohols produced during fermentation can be separated fromthe fermentation media. Any known technique for separating fattyalcohols from aqueous media can be used. One exemplary separationprocess is a two phase (bi-phasic) separation process. This processinvolves fermenting the genetically engineered host cells underconditions sufficient to produce a fatty alcohols, allowing the fattyalcohol to collect in an organic phase, and separating the organic phasefrom the aqueous fermentation broth. This method can be practiced inboth a batch and continuous fermentation processes.

Bi-phasic separation uses the relative immiscibility of fatty alcoholsto facilitate separation. Immiscible refers to the relative inability ofa compound to dissolve in water and is defined by the compound'spartition coefficient. One of ordinary skill in the art will appreciatethat by choosing a fermentation broth and organic phase, such that thefatty alcohol being produced has a high logP value, the fatty alcoholcan separate into the organic phase, even at very low concentrations, inthe fermentation vessel.

The fatty alcohols produced by the methods described herein can berelatively immiscible in the fermentation broth, as well as in thecytoplasm. Therefore, the fatty alcohol can collect in an organic phaseeither intracellularly or extracellularly. The collection of theproducts in the organic phase can lessen the impact of the fatty alcoholon cellular function and can allow the host cell to produce moreproduct.

The methods described herein can result in the production of homogeneouscompounds wherein at least about 60%, 70%, 80%, 90%, or 95% of the fattyalcohols produced will have carbon chain lengths that vary by less thanabout 6 carbons, less than about 4 carbons, or less than about 2carbons. These compounds can also be produced with a relatively uniformdegree of saturation. These compounds can be used directly as fuels,fuel additives, starting materials for production of other chemicalcompounds (e.g., polymers, surfactants, plastics, textiles, solvents,adhesives, etc.), or personal care additives. These compounds can alsobe used as feedstock for subsequent reactions, for example,hydrogenation, catalytic cracking (e.g., via hydrogenation, pyrolisis,or both), to make other products.

In some embodiments, the fatty alcohols produced using methods describedherein can contain between about 50% and about 90% carbon; or betweenabout 5% and about 25% hydrogen. In other embodiments, the fattyalcohols produced using methods described herein can contain betweenabout 65% and about 85% carbon; or between about 10% and about 15%hydrogen.

Surfactant and Detergent Compositions and Bioproducts

The fatty alcohols described herein can be used as or converted into asurfactant or detergent composition. One of ordinary skill in the artwill appreciate that, depending upon the intended purpose of thesurfactant or detergent, different fatty alcohols can be produced andused. For example, when the fatty alcohols described herein are used asa feedstock for surfactant or detergent production, one of ordinaryskill in the art will appreciate that the characteristics of the fattyalcohol feedstock will affect the characteristics of the surfactant ordetergent produced. Hence, the characteristics of the surfactant ordetergent product can be selected for by producing particular fattyalcohols for use as a feedstock.

Bioproducts (e.g., fatty alcohols) comprising biologically producedorganic compounds, particularly fatty alcohols biologically producedusing the fatty acid biosynthetic pathway, have not been produced fromrenewable sources and, as such, are new compositions of matter. Thesenew bioproducts can be distinguished from organic compounds derived frompetrochemical carbon on the basis of dual carbon-isotopic fingerprintingor ¹⁴C dating. Additionally, the specific source of biosourced carbon(e.g., glucose vs. glycerol) can be determined by dual carbon-isotopicfingerprinting (see, e.g., U.S. Pat. No. 7,169,588, which is hereinincorporated by reference).

The ability to distinguish bioproducts from petroleum based organiccompounds is beneficial in tracking these materials in commerce. Forexample, organic compounds or chemicals comprising both biologicallybased and petroleum based carbon isotope profiles may be distinguishedfrom organic compounds and chemicals made only of petroleum basedmaterials. Hence, the instant materials may be followed in commerce onthe basis of their unique carbon isotope profile.

Bioproducts can be distinguished from petroleum based organic compoundsby comparing the stable carbon isotope ratio ¹³C/¹²C) in each fuel. The¹³C/¹²C ratio in a given bioproduct is a consequence of the ¹³C/¹²Cratio in atmospheric carbon dioxide at the time the carbon dioxide isfixed. It also reflects the precise metabolic pathway. Regionalvariations also occur. Petroleum, C₃ plants (the broadleaf), C₄ plants(the grasses), and marine carbonates all show significant differences in¹³C/¹²C and the corresponding δ¹³C values. Furthermore, lipid matter ofC₃ and C₄ plants analyze differently than materials derived from thecarbohydrate components of the same plants as a consequence of themetabolic pathway.

Within the precision of measurement, ¹³C shows large variations due toisotopic fractionation effects, the most significant of which forbioproducts is the photosynthetic mechanism. The major cause ofdifferences in the carbon isotope ratio in plants is closely associatedwith differences in the pathway of photosynthetic carbon metabolism inthe plants, particularly the reaction occurring during the primarycarboxylation (i.e., the initial fixation of atmospheric CO₂). Two largeclasses of vegetation are those that incorporate the “C₃” (orCalvin-Benson) photosynthetic cycle and those that incorporate the “C₄”(or Hatch-Slack) photosynthetic cycle.

In C₃ plants, the primary CO₂ fixation or carboxylation reactioninvolves the enzyme ribulose-1,5-diphosphate carboxylase, and the firststable product is a 3-carbon compound. C₃ plants, such as hardwoods andconifers, are dominant in the temperate climate zones.

In C₄ plants, an additional carboxylation reaction involving anotherenzyme, phosphoenol-pyruvate carboxylase, is the primary carboxylationreaction. The first stable carbon compound is a 4-carbon acid that issubsequently decarboxylated. The CO₂ thus released is refixed by the C₃cycle. Examples of C₄ plants are tropical grasses, corn, and sugar cane.

Both C₄ and C₃ plants exhibit a range of ¹³C/¹²C isotopic ratios, buttypical values are about −7 to about −13 per mil for C₄ plants and about−19 to about −27 per mil for C₃ plants (see, e.g., Stuiver et al.,Radiocarbon 19:355, 1977). Coal and petroleum fall generally in thislatter range. The ¹³C measurement scale was originally defined by a zeroset by Pee Dee Belemnite (PDB) limestone, where values are given inparts per thousand deviations from this material. The “δ¹³C” values areexpressed in parts per thousand (per mil), abbreviated, ‰, and arecalculated as follows:δ¹³C(‰)=[(¹³C/¹²C)_(sample)−(¹³C/¹²C)_(standard)]/(¹³C/¹²C)_(standard)×1000

Since the PDB reference material (RM) has been exhausted, a series ofalternative RMs have been developed in cooperation with the IAEA, USGS,NIST, and other selected international isotope laboratories. Notationsfor the per mil deviations from PDB is δ¹³C. Measurements are made onCO₂ by high precision stable ratio mass spectrometry (IRMS) on molecularions of masses 44, 45, and 46.

The compositions described herein include bioproducts produced by any ofthe methods described herein. Specifically, the bioproduct can have aδ¹³C of about −28 or greater, about −27 or greater, −20 or greater, −18or greater, −15 or greater, −13 or greater, −10 or greater, or −8 orgreater. For example, the bioproduct can have a δ¹³C of about −30 toabout −15, about −27 to about −19, about −25 to about −21, about −15 toabout −5, about −13 to about −7, or about −13 to about −10. In otherinstances, the bioproduct can have a δ¹³C of about −10, −11, −12, or−12.3.

Bioproducts can also be distinguished from petroleum based organiccompounds by comparing the amount of ¹⁴C in each compound. Because ¹⁴Chas a nuclear half life of 5730 years, petroleum based fuels containing“older” carbon can be distinguished from bioproducts which contain“newer” carbon (see, e.g., Currie, “Source Apportionment of AtmosphericParticles”, Characterization of Environmental Particles, J. Buffle andH. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC EnvironmentalAnalytical Chemistry Series (Lewis Publishers, Inc) (1992) 3-74).

The basic assumption in radiocarbon dating is that the constancy of ¹⁴Cconcentration in the atmosphere leads to the constancy of ¹⁴C in livingorganisms. However, because of atmospheric nuclear testing since 1950and the burning of fossil fuel since 1850, ¹⁴C has acquired a second,geochemical time characteristic. Its concentration in atmospheric CO₂,and hence in the living biosphere, approximately doubled at the peak ofnuclear testing, in the mid-1960s. It has since been gradually returningto the steady-state cosmogenic (atmospheric) baseline isotope rate(¹⁴C/¹²C) of about 1.2×10⁻¹², with an approximate relaxation “half-life”of 7-10 years. (This latter half-life must not be taken literally;rather, one must use the detailed atmospheric nuclear input/decayfunction to trace the variation of atmospheric and biospheric ¹⁴C sincethe onset of the nuclear age.)

It is this latter biospheric ¹⁴C time characteristic that holds out thepromise of annual dating of recent biospheric carbon. ¹⁴C can bemeasured by accelerator mass spectrometry (AMS), with results given inunits of “fraction of modern carbon” (f_(M)). f_(M) is defined byNational Institute of Standards and Technology (NIST) Standard ReferenceMaterials (SRMs) 4990B and 4990C. As used herein, “fraction of moderncarbon” or “f_(M)” has the same meaning as defined by National Instituteof Standards and Technology (NIST) Standard Reference Materials (SRMs)4990B and 4990C, known as oxalic acids standards HOxI and HOxII,respectively. The fundamental definition relates to 0.95 times the¹⁴C/¹²C isotope ratio HOxI (referenced to AD 1950). This is roughlyequivalent to decay-corrected pre-Industrial Revolution wood. For thecurrent living biosphere (plant material), f_(M) is approximately 1.1.

The compositions described herein include bioproducts that can have anf_(M) ¹⁴C of at least about 1. For example, the bioproduct can have anf_(M) ¹⁴C of at least about 1.01, an f_(M) ¹⁴C of about 1 to about 1.5,an f_(M) ¹⁴C of about 1.04 to about 1.18, or an f_(M) ¹⁴C of about 1.111to about 1.124.

Another measurement of ¹⁴C is known as the percent of modern carbon,pMC. For an archaeologist or geologist using ¹⁴C dates, AD 1950 equals“zero years old”. This also represents 100 pMC. “Bomb carbon” in theatmosphere reached almost twice the normal level in 1963 at the peak ofthermo-nuclear weapons. Its distribution within the atmosphere has beenapproximated since its appearance, showing values that are greater than100 pMC for plants and animals living since AD 1950. It has graduallydecreased over time with today's value being near 107.5 pMC. This meansthat a fresh biomass material, such as corn, would give a ¹⁴C signaturenear 107.5 pMC. Petroleum based compounds will have a pMC value of zero.Combining fossil carbon with present day carbon will result in adilution of the present day pMC content. By presuming 107.5 pMCrepresents the ¹⁴C content of present day biomass materials and 0 pMCrepresents the ¹⁴C content of petroleum based products, the measured pMCvalue for that material will reflect the proportions of the twocomponent types. For example, a material derived 100% from present daysoybeans would give a radiocarbon signature near 107.5 pMC. If thatmaterial was diluted 50% with petroleum based products, it would give aradiocarbon signature of approximately 54 pMC.

A biologically based carbon content is derived by assigning “100%” equalto 107.5 pMC and “0%” equal to 0 pMC. For example, a sample measuring 99pMC will give an equivalent biologically based carbon content of 93%.This value is referred to as the mean biologically based carbon resultand assumes all the components within the analyzed material originatedeither from present day biological material or petroleum based material.

A bioproduct described herein can have a pMC of at least about 50, 60,70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100. In other instances, abioproduct described herein can have a pMC of between about 50 and about100; about 60 and about 100; about 70 and about 100; about 80 and about100; about 85 and about 100; about 87 and about 98; or about 90 andabout 95. In yet other instances, a bioproduct described herein can havea pMC of about 90, 91, 92, 93, 94, or 94.2.

Fuel additives are used to enhance the performance of a fuel or engine.For example, fuel additives can be used to alter the freezing/gellingpoint, cloud point, lubricity, viscosity, oxidative stability, ignitionquality, octane level, and/or flash point. In the United States, allfuel additives must be registered with Environmental Protection Agency.The names of fuel additives and the companies that sell the fueladditives are publicly available by contacting the EPA or by viewing theagency's website. One of ordinary skill in the art will appreciate thatthe fatty alcohol-based biofuels described herein can be mixed with oneor more fuel additives to impart a desired quality.

The fatty alcohol-based surfactants and/or detergents described hereincan be mixed with other surfactants and/or detergents well known in theart.

In some examples, the mixture can include at least about 10%, 15%, 20%,30%, 40%, 50%, or 60% by weight of the fatty alcohol. In other examples,a surfactant or detergent composition can be made that includes at leastabout 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% of afatty alcohol that includes a carbon chain that is 8, 10, 12, 13, 14,15, 16, 17, 18, 19, 20, 21 or 22 carbons in length. Such surfactant ordetergent compositions can additionally include at least one additiveselected from a surfactant; a microemulsion; at least about 5%, 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% of surfactantor detergent from nonmicrobial sources such as plant oils or petroleum.

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only. They are not to beconstrued as limiting the scope or content of the invention in any way.

EXAMPLES Example 1 Identification of Carboxylic Acid Reductase (CAR)Homologs

The carboxylic acid reductase (CAR) from Nocardia sp. strain NRRL 5646can reduce carboxylic acids (e.g., fatty acids) into their correspondingaldehydes without utilizing separate activating enzymes, such asacyl-CoA synthases (Li et al., J. Bacteriol. 179:3482-3487, 1997; He etal., Appl. Environ. Microbiol. 70:1874-1881, 2004)).

A BLAST search using the NRRL 5646 CAR amino acid sequence (Genpeptaccession AAR91681) (SEQ ID NO:16) as the query sequence identifiedapproximately 20 homologous sequences. Three homologs, listed in Table7, were evaluated for their ability to convert fatty acids into fattyaldehydes in vivo when expressed in E. coli.

At the nucleotide sequence level, carA (SEQ ID NO:19), carB (SEQ IDNO:21), and fadD9 (SEQ ID NO:17) demonstrated 62.6%, 49.4%, and 60.5%homology, respectively, to the car gene (AY495697) of Nocardia sp. NRRL5646 (SEQ ID NO:15). At the amino acid level, CARA (SEQ ID NO:20), CARB(SEQ ID NO:22), and FadD9 (SEQ ID NO:18) demonstrated 62.4%, 59.1% and60.7% identity, respectively, to CAR of Nocardia sp. NRRL 5646 (SEQ IDNO:16).

TABLE 7 CAR-like Protein and the corresponding coding sequences. GenpeptGene accession Locus_tag Annotation in GenBank name NP_217106 Rv 2590Probable fatty-acid-CoA ligase fadD9 (FadD9) ABK75684 MSMEG NADdependent carA 2956 epimerase/dehydratase family protein YP_889972.1MSMEG NAD dependent carB 5739 epimerase/dehydratase family protein

Example 2 Identification of Alcohol Dehydrogenase Genes

Reverse Engineering

E. coli contains at least one enzyme that catalyzes the reversibleoxidoreduction of fatty aldehydes and fatty alcohols (i.e. fattyaldehyde reductase/alcohol dehydrogenase). Reverse engineering was usedto identify such fatty aldehyde reducatases/fatty alcohol dehydrogensesin E. coli MG1655 cells expressing the acyl-ACP reductase YP_(—)400611from Synechococcus elongatus (Synpcc7942 1594) (SEQ ID NO:196). Four 3ml, LB cultures were grown overnight at 37° C., and 55 mL of stationaryphase cultures were used to inoculate four independent 5.5 mL of LB.Those 5.5 mL cultures were then grown to an OD₆₀₀ of 0.8-1.0 and werethen used to inoculate a corresponding number of 2 L baffledshakeflasks, each with 500 mL Hu-9 minimal media. 20 hrs after inductionthe cells were pelleted at 4,000×g for 20 min. The cell pellet wasresuspended in 30 mL of 100 mM phosphate buffer at pH 7.2 with 1×Bacterial Protease Arrest (G Biosciences). The cells were lysed in afrench press at 15,000 psi with two passes through the instrument. Thecell debris was then removed by centrifuging at 10,000×g for 20 mins.The cell lysate was loaded onto two HiTrapQ columns (GE Healthcare)connected in series. The following buffers were used to elute proteins:(A) 50 mM Tris, pH 7.5 and (B) 50 mM Tris, pH 7.5 with 1 M NaCl. Agradient from 0% B to 100% B was run over 5 column volumes at a flowrate of 3 mL/min while 4 mL fractions were collected.

The fractions were assayed for alcohol dehydrogenase activity by taking190 μL of a protein fraction and adding 5 μL of a 20 mM NADPH (Sigma)solution and 5 μL of a 20 mM dodecanal (Fluka) solution in DMSO. Thereactions were incubated at 37° C. for 1 hr. They were then extractedwith 100 μL of ethyl acetate and analyzed for dodecanol via GC/MS.Fractions eluting around 350 mM NaCl contained alcohol dehydrogenaseactivity.

Fractions containing alcohol dehydrogenase activity were pooled andloaded onto a 1 mL ResourceQ column (GE Healthcare). The same conditionsused for the HiTrapQ column were used, except 0.5 mL fractions werecollected. Protein fractions demonstrating alcohol dehydrogenaseactivity were then pooled and concentrated using Amicon (Milipore)protein concentrators (10,000 kDa cutoffs) to a volume of 1 mL. Thesolution was then loaded onto a HiPrep 200 size exclusion column (GEHealthcare). A buffer solution containing 50 mM Tris, pH 7.5, and 150 mMNaCl was run through the column at a rate of 0.3 mL per min. 2 mLfractions were collected. Two protein fractions contained alcoholdehydrogenase activity. These two fractions, plus fractions before andafter these two fractions, were loaded onto a polyacrylamide gel andstained with SimplySafe Commassie stain (Invitrogen).

Comparing the bands in the active and inactive fractions, one proteinband appeared in the active fractions that was not seen in the inactivefraction. This protein band was cut from gel and submitted to theStanford Mass Spectroscopy Facility for LC/MS/MS protein sequencing. Oneof the proteins identified in this analysis was YahK.

To verify that YahK was indeed an alcohol dehydrogenase, yahK wasknocked out in E. coli MG1655(DE3, ΔfadD, ΔyjgB) (control strain)(described in Example 4). The yahK knock-out strain MG1655(DE3, ΔfadD,Δyjg,B ΔyahK) was constructed using the lamda red system (described inExample 4) with the following primers:

yahK_F (SEQ ID NO: 197)(CATATCAGGCGTTGCCAAATACACATAGCTAATCAGGAGTAAACACAAT G) and yahK_R (SEQ IDNO: 198) (AATCGCACACTAACAGACTGAAAAAATTAATAAATACCCTGTGGTTTAA C).This ΔyahK strain and the control strain, both expressing the acyl-ACPreductase YP_(—)400611, were cultured under conditions described above.Cell free lysates were made from both strains, and each lysate wasassayed for alcohol dehydrogenase activity as discussed above.

The ΔyahK strain did not convert dodecanal to dodecanol, while the wildtype strain had this activity. For additional verification, each lysatewas run on a HiTrapQ column as described above. The wild type lysate hadalcohol dehydrogenase activity in fractions eluting around 350 mM NaCl,while the ΔyahK lysate had no alcohol dehydrogenase activity in thisregion.

Bioinformatics

It was reasoned that possible alcohol dehydrogenases in E. coli weremembers of four protein families: Zn-dependent alcohol dehydrogenases(Pfam 00107 and 08240), Fe-dependent alcohol dehydrogenases (Pfam00465), aldo-keto reductases (Pfam 00248) and short-chain dehydrogenases(Pfam 00106) (Pfam=protein family according to “pfam.sanger.ac.uk”).Using the Pfam motifs, all members of these four protein families in E.coli were identified (listed in FIG. 10). From this list, the following8 candidates were chosen for experimental analysis: yahK, yjgB, adhP,dkgA, dkgB, yhdH, ydjL, and yqhD.

To determine if these genes could reduce fatty aldehydes to fattyalcohols, these 8 genes were cloned into a pET-Duet vector along with E.coli 'tesA. These genes were then transformed into E. coli (DE3) MG1655ΔyjgBΔyahK cells. Next 3 mL overnight starter cultures were grown in LBwith carbanecillin (100 mg/L) at 37° C. A control strain lacking acandidate alcohol dehydrogenase was also included in the experiment. 1mL of each overnight culture was used to inoculate 50 mL of fresh LBwith carbanecillin. The cultures were shaken at 37° C. until reaching anOD₆₀₀ of 0.8-1. The cultures were then transferred to 18° C., inducedwith 1 mM IPTG, and shaken overnight.

Cell free lysates were prepared by centrifuging the cultures at 4,000×gfor 20 mins. The cultures were then resuspended in 1 mL of Bugbuster(Novagen) and gently shaken at room temperature for 5 min. The celldebris was removed by spinning at 15,000×g for 10 min. The resultinglysates were assayed for alcohol dehydrogenase activity by mixing 88 μLof lysate, 2 μL of 40 mM cis-11-hexadecenal in DMSO, and 10 μL of 20 mMNADPH. The samples were incubated at 37° C. for 30 min. and were thenextracted with 100 μL of ethyl acetate. The extracts were analyzed usingGC/MS.

All proteins showed significantly better conversion ofcis-11-hexadecenal to cis-11-hexadecanol as compared with the 'TesA onlycontrol (see Table 8). These results were confirmed in assays usingdodecanal instead of cis-11-hexadecenal as the substrate (see Table 8).

To investigate how these enzymes contribute to fatty alcoholdehydrogenase activity in E. coli under production conditions, first theyjgB yahK double knock-out strain in MG1655(DE3, ΔfadD) (describedabove) was tested by transforming it with a plasmid expressing acyl-ACPreductase YP_(—)400611 and analyzing fatty aldehyde and fatty alcoholtiters. The test strain also contained a plasmid expressing adecarbonylase. This double knock-out mutant showed slightly higher fattyaldehyde titers in several experiments (see, e.g., FIG. 11), confirmingthat these two putative alcohol dehydrogenases contribute to fattyalcohol dehydrogenase activity in E. coli under production conditions(see also Example 4 for similar results from a MG1655(DE3, ΔfadD ΔyjgB)strain). Next, two additional genes, yncB and ydjA, were deleted in theyjgB yahK double mutant. YdjA, which is not a member of the four proteinfamilies mentioned above, demonstrated slightly elevated fatty aldehydelevels (see FIG. 11), indicating that it may also contribute to fattyalcohol dehydrogenase activity in E. coli under production conditions.

Additionally, the active fatty alcohol dehydrogenases from Table 8 werealso deleted in MG1655 (DE3, ΔfadD, Δyjg,B ΔyahK) and tested asdescribed above. Several of these deletion strains showed slightlyelevated fatty aldehyde levels, suggesting that these may alsocontribute to fatty alcohol dehydrogenase activity in E. coli underproduction conditions (see FIG. 12).

TABLE 8 Overexpression of putative fatty alcohol dehydrogenase genesGC/MS Assay NADPH assay % conversion to initial corresponding alcoholrate (slope) substrate dodecanal cis 11-hexadecenal cis 11-hexadecenalOverexpression: none 9 12 0.2 YjgB 54 89 24.8 YahK 47 87 28.3 AdhP 52 454.1 YdjL 51 23 0.14 YhdH 59 74 13.7 YqhD 55 23 7.3 yafB (dkgB) 52 65 9.4YqhE (dkgA) 45 50 9.6

Example 3 Expression of CAR Homologs and Alcohol Dehydrogenase in E.coli

A. CAR Plasmid Construction

Three E. coli expression plasmids were constructed to express the genesencoding the CAR homologs listed in Table 7. First, fadD9 was amplifiedfrom genomic DNA of Mycobacterium tuberculosis H37Rv (obtained from TheUniversity of British Columbia, and Vancouver, BC Canada) using theprimers fadD9F and FadDR (see Table 9). The PCR product was first clonedinto PCR-blunt (Invitrogen) and then released as an NdeI-AvrII fragment.The NdeI-AvrII fragment was then cloned between the NdeI and AvrII sitesof pACYCDuet-1 (Novogen) to generate pACYCDuet-1-fadD9.

The carA gene was amplified from the genomic DNA of Mycobacteriumsmegmatis MC2 155 (obtained from the ATCC (ATCC 23037D-5)) using primersCARMCaF and CARMCaR (see Table 9). The carB gene was amplified from thegenomic DNA of Mycobacterium smegmatis MC2 155 (obtained from the ATCC(ATCC 23037D-5)) using primers CARMCbF and CARMCbR (see Table 9). EachPCR product was first cloned into PCR-blunt and then released as anNdeI-AvrII fragment. Each of the two fragments was then subclonedbetween the NdeI and AvrII sites of pACYCDuet-1 (Novogen) to generatepACYCDuet-1-carA and pACYCDuet-1-carB.

TABLE 9 Primers used to amplify genes encoding CAR homologs fadD9F catATGTCGATCAACGATCAGCGACTGAC (SEQ ID NO: 1) fadD9R cctaggTCACAGCAGCCCGAGCAGTC (SEQ ID NO: 2) CARMCaF cat ATGACGATCGAAACGCG (SEQID NO: 3) CARMCaR cctagg TTACAGCAATCCGAGCATCT (SEQ ID NO: 4) CARMCbF catATGACCAGCGATGTTCAC (SEQ ID NO: 5) CARMCbR cctagg TCAGATCAGACCGAACTCACG(SEQ ID NO: 6)B. Alcohol Dehydrogenase Plasmid Construction

The plasmid pETDuet-1-'tesA-yjgB carries 'tesA and yjgB (a putativealcohol dehydrogenase; GenBank accession number, NP_(—)418690; GenPeptaccession number AAC77226) from the E. coli K12 strain.

The gene yjgB (GenBank accession number, NP_(—)418690) was amplifiedfrom the genomic DNA of E. coli K-12 using the following primers.

The yjgB insert was generated by PCR using the following primers:

NcoI YjgB forward: (SEQ ID NO: 199)aatccTGGCATCGATGATAAAAAGCTATGCCGCAAAAG HindIII YjgB reverse: (SEQ ID NO:200) ataaaagctTTCAAAAATCGGCTTTCAACACCACGCGGThe PCR product was then subcloned into the NcoI and HindIII sites ofpETDuet-1-'tesA to generate pETDuet-1-'tesA-yjgB.

The plasmid pETDuet-1-'tesA-alrAadp1 carries 'tesA and alrAadp1 (GenPeptaccession number CAG70248.1) from Acinetobacter baylyi ADP1.

The gene alrAadp1 was amplified from the genomic DNA of Acinetobacterbaylyi ADP1 by a two-step PCR procedure. The first set of PCR reactionseliminated an internal NcoI site at by 632-636 with the following primerpairs:

ADP1 Alr mut1 reverse: (SEQ ID NO: 201)5′-GACCACGTGATCGGCCCCCATAGCTTTGAGCTCATC ADP1 Alr1 mut1 forward: (SEQ IDNO: 202) 5′-GATGAGCTCAAAGCTATGGGGGCCGATCACGTGGTCThe PCR products were then isolated, purified using the Qiagen gelextraction kit, and used as inputs for a second PCR reaction with thefollowing primers to produce full-length AlrAadp1 with a C→T mutation atposition 633:

NcoI ADP1 Alr1 forward: (SEQ ID NO: 203)5′-AATACCATGGCAACAACTAATGTGATTCATGCTTATGCTGCA HindIII ADP1 Alr1 reverse:(SEQ ID NO: 204) 5′-ATAAAAGCTTTTAAAAATCGGCTTTAAGTACAATCCGATAACThe plasmid pETDuet-1-'tesA-alrAadp1 was prepared by inserting thealrAadp1 gene (gene locus-tag=“ACIAD3612”), a homolog of Acinetobacterbaylyi ADP1, into the NcoI and HindIII sites of pETDuet-1-'tesA.B. Evaluation of Fatty Aldehyde and Fatty Alcohol Production

In order to evaluate the affect of carboxylic acid reductases andalcohol dehydrogenases on the production of fatty alcohols, variouscombinations of the prepared plasmids were transformed in the E. colistrain C41 (DE3, ΔfadE) (described in PCT/US08/058788).

For example, the plasmid pACYCDuet-1-carA, encoding the CAR homologcarA, was co-transformed with pETDuet-1-'tesA-alrAadp1 (see, e.g., FIG.1).

The plasmid pACYCDuet-1-carB, encoding the CAR homolog carB, wasco-transformed with pETDuet-1-'tesA. In addition, pACYCDuet-1-carB wasalso separately co-transformed with pETDuet-1-'tesA-yjgB andpETDuet-1-'tesA-alrAadp1. As a control, pACYCDuet-1-carB wasco-transformed with the empty vector pETDuet-1 (see, e.g., FIG. 1).

The plasmid pACYCDuet-1-fadD9, encoding the CAR homolog fadD9, wasco-transformed with pETDuet-1-'tesA. In addition, pACYCDuet-1-fadD9 wasalso separately co-transformed with pETDuet-1-'tesA-yjgB andpETDuet-1-'tesA-alrAadp1. As a control, pACYCDuet-1-fadD9 wasco-transformed with the empty vector pETDuet-1 (see, e.g., FIG. 1).

As an additional control, pETDuet-1-'tesA-yjgB was co-transformed withthe empty vector pACYCDuet-1.

The E. coli transformants were grown in 3 mL of LB medium supplementedwith carbenicillin (100 mg/L) and chloramphenicol (34 mg/L) at 37° C.After overnight growth, 15 μL of culture was transferred into 2 mL offresh LB medium supplemented with carbenicillin and chloramphenicol.After 3.5 hours of growth, 2 mL of culture were transferred into a 125mL flask containing 20 mL of M9 medium with 2% glucose and withcarbenicillin and chloramphenicol. When the OD₆₀₀ of the culture reached0.9, 1 mM of IPTG was added to each flask. After 20 hours of growth at37° C., 20 mL of ethyl acetate (with 1% of acetic acid, v/v) was addedto each flask to extract the fatty alcohols produced during thefermentation. The crude ethyl acetate extract was directly analyzed withGC/MS as described herein.

The measured retention times were 6.79 minutes for cis-5-dodecen-1-ol,6.868 minutes for 1-dodecanol, 8.058 minutes for cis-7-tetradecen-1-ol,8.19 minutes for 1-tetradecanol, 9.208 minutes for cis-9-hexadecen-1-ol,9.30 minutes for 1-hexadecanol, and 10.209 minutes forcis-11-octadecen-1-ol.

The co-expression of the leaderless tesA and any of the three car genesin E. coli resulted in high titers of fatty alcohols and detectablefatty aldehyde production (FIGS. 1, 2, 5). The expression of carA orcarB with the leaderless tesA and alrAadp1 resulted in fatty alcoholtiters of greater than 700 mg/L and reduced fatty aldehyde production.Likewise, fadD9 co-expressed with the leaderless tesA and alrAadp1produced over 300 mg/L of fatty alcohol. When expressed without theleaderless tesA, neither carB nor fadD9 produced more than 10 mg/L offatty alcohols (possibly resulting from the accumulation of free fattyacids in the cell due to endogenous tesA). Taken together, this dataindicates that fatty acids are the substrates for these CAR homologs andthat overexpression of a thioesterase, such as 'tesA (to release fattyacids from acyl-ACP), achieves significant production of fatty alcohols.

In one fermentation, E. coli strain C41 (DE3, ΔfadE) co-transformed withpACYCDuet-1-carB+pETDuet-1-tesA produced an average of 695 mg/L of fattyalcohols and 120 mg/L of fatty aldehydes. The presence of large amountsof fatty aldehydes is consistent with CAR being an aldehyde-generating,fatty acid reductase (AFAR). This mechanism is different fromalcohol-generating fatty acyl-CoA reductases (FAR), represented byJjFAR, and fatty acyl-CoA reductases, represented by Acr1.

The production of fatty alcohols from fatty aldehydes in the E. colistrains described above may have been catalyzed by an endogenous alcoholdehydrogenase(s). E. coli produces an alcohol dehydrogenase(s) (e.g.,yjgB) capable of converting fatty aldehydes of various chain-length intofatty alcohols (Naccarato et al., Lipids 9: 419-428 (1974); Reiser etal., J. Bacteriol. 179: 2969-2975 (1997); Venkitasubramanian et al., J.Biol. Chem. 282:478-485 (2007)).

Therefore, alcohols dehydrogenases may also play a role in the fattyalcohol biosynthetic pathway in addition to carboxylic acid reductases.For example, expression of either yjgB or alrAadp1 with carB and theleaderless tesA significantly reduced the accumulation of fattyaldehydes, compared to strains that did not overexpress yjgB or alrAadp1(FIG. 2).

Following the fermentations where pACYCDuet-1-carB was transformed in E.coli strain C41 (DE3, ΔfadE), a white, round, disk-like deposit wasobserved at the bottom center of the flasks used for fatty alcoholproduction with recombinant E. coli strains. In contrast, no suchdeposits were observed at the bottom of the control flasks that did notexpress car homologs. GC/MS analysis of the deposit dissolved in ethylacetate (with 1% of acetic acid, v/v) revealed that the deposit was afatty alcohol deposit.

C. Types of Fatty Alcohols Produced by Different CAR Homologs

Depending upon the CAR homolog expressed in E. coli strain C41 (DE3,ΔfadE), different mixtures of fatty alcohols were produced. Differentcompositions of fatty alcohols were observed among the three CARhomologs evaluated (see Table 10). FadD9 produced more C₁₂ fattyalcohols relative to other fatty alcohols with carbon chain lengthsgreater than 12. Both CarA and CarB produced a wider range in chainlength of fatty alcohols than was observed when expressing FadD9.

TABLE 10 Acyl-composition of fatty alcohols produced by recombinant E.coli strains Expressed with TesA* Acyl-composition of fatty alcohols (%)and AlrAadp1 C10:0 C12 C14:1 C14:0 C16:1 C16:0 C18:1 CarA trace 38 13 2716 4 3 FadD9 trace 63 14 16 7 trace trace CarB trace 32 11 41 12 tracetrace *the leaderless TesA. C12, including C12:0 and C12:1 fattyalcohol.D. Quantification and Identification of Fatty Alcohols

GC-MS was performed using an Agilent 5975B MSD system equipped with a 30m×0.25 mm (0.10 μm film) DB-5 column. The column temperature was 3 minisothermal at 100° C. The column was programmed to rise from 100° C. to320° C. at a rate of 20° C./min. When the final temperature was reached,the column remained isothermal for 5 minutes at 320° C. The injectionvolume was 1 pt. The carrier gas, helium, was released at 1.3 mL/min.The mass spectrometer was equipped with an electron impact ionizationsource. The ionization source temperature was set at 300° C.

Prior to quantification, various alcohols were identified using twomethods. First, the GC retention time of each compound was compared tothe retention time of a known standards, such as cetyl alcohol,dodecanol, tetradecanol, octadecanol, and cis-9-octadecenol. Second,identification of each compound was confirmed by matching the compound'smass spectrum to a standard's mass spectrum in the mass spectra library(e.g., C12:0, C12:1, C13:0, C14:0, C14:1, C15:0. C16:0, C16:1, C17:0,C18:0 and C18:1 alcohols).

Example 4 Production of Fatty Alcohol by Heterologous Expression of CARHomologs in E. coli MG1655 (DE3, ΔfadD)

Construction of fadD Deletion Strain

The fadD gene of E. coli MG1655 was deleted using the lambda red system(Datsenko et al., 2000, Proc. Natl. Acad. Sci. USA. 97: 6640-6645) asfollows:

The chloramphenicol acetyltransferase gene from pKD3 was amplified withthe primers fad1 (5′-TAACCGGCGTCTGACGACTGACTTAACGCTCAGGCTTTATTGTCCACTTTGTGTAGGCTGGAGCTGCTTCG-3′) (SEQ ID NO:205), and fad2(5′-CATTTGGGGTTGCGATGACGACGAACACGCATTTTAGAGGTGAAGAATTGCATATGAATATCCTCCTTTAGTTCC-3′) (SEQ ID NO:206).This PCR product was electroporated into E. coli MG1655 (pKD46). Thecells were plated on L-chloramphenicol (30 μg/mL) (L-Cm) and grownovernight at 37° C. Individual colonies were picked on to another L-Cmplate and grown at 42° C. These colonies were then patched to L-Cm andL-carbenicillin (100 mg/mL) (L-Cb) plates and grown at 37° C. overnight.Colonies that were Cm^(R) and Cb^(S) were evaluated further by PCR toensure the PCR product inserted at the correct site. PCR verificationwas performed on colony lysates of these bacteria using the primers fadF(5′-CGTCCGTGGTAATCATTTGG-3′) (SEQ ID NO:207) and fadR(5′-TCGCAACCTTTTCGTTGG-3′) (SEQ ID NO:208). Expected size of theΔfadD::Cm deletion was about 1200 by (FIG. 4). The chloramphenicolresistance gene was eliminated using a FLP helper plasmid as describedin Datsenko et al., Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000). PCRverification of the deletion was performed with primers fadF and fadR(FIG. 4). The MG1655 ΔfadD strain was unable to grow on M9+ oleate agarplates (oleate as carbon source). It was also unable to grow in M9+oleate liquid media. The growth defect was complemented by an E. colifadD gene supplied in trans (in pCL1920-Ptrc).Construction of MG1655(DE3, ΔfadD) Strain

To generate a T7-responsive strain, the λDE3 Lysogenization Kit(Novagen) was utilized, which is designed for site-specific integrationof λDE3 prophage into an E. coli host chromosome, such that thelysogenized host can be used to express target genes cloned in T7expression vectors. λDE3 is a recombinant phage carrying the cloned genefor T7 RNA polymerase under lacUV5 control. Briefly, the host strain wascultured in LB supplemented with 0.2% maltose, 10 mM MgSO₄, andantibiotics at 37° C. to an OD₆₀₀ of 0.5. Next, 10⁸ pfu λDE3, 10⁸ pfuHelper Phage, and 10⁸ pfu Selection Phage were incubated with 10 μL hostcells. The host/phage mixture was incubated at 37° C. for 20 min toallow phage to adsorb to host. Finally, the mixture was pipeted onto anLB plate supplemented with antibiotics. The mixture was spread evenlyusing plating beads, and the plates were inverted plates and incubatedat 37° C. overnight.

λDE3 lysogen candidates were evaluated by their ability to support thegrowth of the T7 Tester Phage. T7 Tester Phage is a T7 phage deletionmutant that is completely defective unless active T7 RNA polymerase isprovided by the host cell. The T7 Tester Phage makes very large plaqueson authentic λDE3 lysogens in the presence of IPTG, while much smallerplaques are observed in the absence of inducer. The relative size of theplaques in the absence of IPTG is an indication of the basal levelexpression of T7 RNA polymerase in the lysogen, and can vary widelybetween different host cell backgrounds.

The following procedure was used to determine the presence of DE3lysogeny. First, candidate colonies were grown in LB supplemented with0.2% maltose, 10 mM MgSO₄, and antibiotics at 37° C. to an OD₆₀₀ of 0.5.An aliquot of T7 Tester Phage was then diluted in 1× Phage DilutionBuffer to a titer of 2×10³ pfu/mL. In duplicate tubes, 100 μL host cellswere mixed with 100 μL diluted phage. The host/phage mixture wasincubated at room temperature for 10 min to allow phage to adsorb tohost. Next, 3 mL of molten top agarose was added to each tube containinghost and phage. The contents of one duplicate were plated onto an LBplate and the other duplicate onto an LB plate supplemented with 0.4 mMIPTG (isopropyl-b-thiogalactopyranoside) to evaluate induction of T7 RNApolymerase. Plates were allowed to sit undisturbed for 5 min until thetop agarose hardened. The plates were then inverted at 30° C. overnight.

Construction of MG1655(DE3, ΔfadD, yjgB::kan) Strain

The yjgB knockout strain, MG1655(DE3, ΔfadD, yjgB::kan), was constructedby using the following lambda red system (Datsenko et al., Proc. Natl.Acad. Sci. USA 97:6640-6645 (2000)):

The kanamycin resistant gene from pKD13 was amplified with the primersyjgBRn (5′-GCGCCTCAGATCAGCGCTGCGAATGATTTTCAAAAATCGGCTTTCAACACTGTAGGCTGGAGCTGCTTCG-3′) (SEQ ID NO:209), and yjgBFn(5′-CTGCCATGCTCTACACTTCCCAAACAACACCAGAGAAGGACCAAAAAATGATTCCGGGGATCCGTCGACC-3′) (SEQ ID NO:210). The PCR product was thenelectroporated into E. coli MG1655(DE3, ΔfadD)/pKD46. The cells wereplated on kanamycin (50 μg/mL) (L-Kan) and grown overnight at 37° C.Individual colonies were picked on to another L-Kan plate and grown at42° C. These colonies were then patched to L-Kan and carbenicillin (100mg/mL) (L-Cb) plates and grown at 37° C. overnight. Colonies that werekan^(R) and Cb^(S) were evaluated further by PCR to ensure the PCRproduct was inserted at the correct site. PCR verification was performedon colony lysates of these bacteria using the primers BF(5′-gtgctggcgataCGACAAAACA-3′) (SEQ ID NO:211) and BR(5′-CCCCGCCCTGCCATGCTCTACAC-3′) (SEQ ID NO:212). The expected size ofthe yjgB::kan knockout was about 1450 bp.

Evaluation of FadD on Fatty Alcohol Production Using MG1655(DE3, ΔfadD)Strain

In Example 3, a fadE deletion strain was used for fatty aldehyde andfatty alcohol production from 'TesA, CAR homologs, and endogenousalcohol dehydrogenase(s) in E. coli. To demonstrate that CAR homologsused fatty acids instead of acyl-CoA as a substrate, the gene encodingfor acyl-CoA synthase in E. coli (fadD) was deleted so that the fattyacids produced were not activated with CoA. E. coli strain MG1655(DE3,ΔfadD) was transformed with pETDuet-1-'tesA and pACYCDuet-1-carB. Thetransformants were evaluated for fatty alcohol production using themethods described herein. These transformants produced about 360 mg/L offatty alcohols (dodecanol, dodecenol, tetredecanol, tetredecenol, cetyl,hexadecenol, and octadecenol).

YjgB is an Alcohol Dehydrogenase

To confirm that YjgB was an alcohol dehydrogenase responsible forconverting fatty aldehydes into their corresponding fatty alcohols,pETDuet-1-'tesA and pACYCDuet-1-fadD9 were co-transformed into eitherMG1655(DE3, ΔfadD) or MG1655(DE3, ΔfadD, yjgB::kan). At the same time,MG1655(DE3, ΔfadD, yjgB::kan) was transformed with bothpETDuet-1-'tesA-yjgB and pACYCDuet-1-fadD9.

The E. coli transformants were grown in 3 mL of LB medium supplementedwith carbenicillin (100 mg/L) and chloramphenicol (34 mg/L) at 37° C.After overnight growth, 15 μL of culture was transferred into 2 mL offresh LB medium supplemented with carbenicillin and chloramphenicol.After 3.5 hrs of growth, 2 mL of culture was transferred into a 125 mLflask containing 20 mL of M9 medium with 2% glucose, carbenicillin, andchloramphenicol. When the OD₆₀₀ of the culture reached 0.9, 1 mM of IPTGwas added to each flask. After 20 hrs of growth at 37° C., 20 mL ofethyl acetate (with 1% of acetic acid, v/v) was added to each flask toextract the fatty alcohols produced during the fermentation. The crudeethyl acetate extract was directly analyzed with GC/MS as describedherein.

The yjgB knockout strain resulted in significant accumulation ofdodecanal and a lower fatty alcohol titer (FIG. 5). The expression ofyjgB from plasmid pETDuet-1-'tesA-yjgB in the yjgB knockout straineffectively removed the accumulation of dodecanal (FIG. 5). The datashows that YjgB was involved in converting dodecanal into dodecanol andthat there may be other alcohol dehydrogenase(s) present in E. coli toconvert other aldehydes into alcohols. Dodecanal accumulated in the yjgBknockout strain, but it was not observed in either the wild-type strain(MG1655(DE3, ΔfadD)) or the yjgB knockout strain with the yjgBexpression plasmid. The arrows (in FIG. 5) indicate the GC trace ofdodecanal (C12:0 aldehyde).

Example 5 Production of Saturated Fatty Alcohols in E. coli

Fatty alcohols for commercial uses are saturated. However, E. colitypically has a certain amount (about 20-25%) of unsaturated fatty acidsin its membrane to maintain fluidity. An E. coli strain was engineeredthat was able to produce exclusively saturated fatty acids in a mediumnot supplemented with unsaturated fatty acid or cyclopropane-fatty acidand was able to produce saturated fatty alcohols.

Two enzymes, a dehydratase/isomerase and a ketoacylsynthase I (KASI),encoded by fabA and fabB, respectively, are involved in unsaturatedfatty acid biosynthesis. Usually, an E. coli strain lacking either FabAor FabB does not survive without supplementation of unsaturated fattyacids, such as oleate. To overcome this, the fabB gene was knocked outof an E. coli host strain, and the strain was able to grow withoutunsaturated fatty acid supplementation by genetically engineering thecells to express a recombinant desaturase gene (AF037430, encoding DesA)from Bacillus subtilis. Although the first generation of the strainexpressing desA required oleate for normal growth, subsequent plating ofthe strain on L Agar plates several times resulted in a strain that didnot require oleate for growth.

Materials

E. coli JWC280 cells (described in Campbell et al., Mol. Microbiol.47:793-805 (2003)) and E. coli GRT23 cells (described in Morgan-Kiss etal., Arch. Microbiol. 190:427-437 (2008)) were obtained from JohnCronan.

Plasmid Construction

The desA gene (also referred to as Δ5 des) was amplified with primersdelta5Fn and delta5Rn (listed in Table 11) from the genomic DNA ofBacillus subtilis str. 168 and digested with AvrII and EcoRI. The desAgene was then cloned into pET-21(a), which had been linearized withAvrII-EcoRI, to produce pET-21a-Δ5. The desA gene was then removed as anNdeI-EcoRI fragment from pET-21a-Δ5 and inserted between the NdeI andEcoRI sites of OP180, a pACYC derived plasmid carrying a trc promoter.The resultant plasmid was named pACYC-Δ5.

A desA_kan gene cassette was cloned between the AvrII-BamHI sites ofCDFDuet-1. A kan gene cassette was produced by EcoRI and BamHI digestionof a PCR product that was amplified with primers kanF and kanR (seeTable 11) from pKD13 as the template (pKD13 was obtained from The ColiGenetic Stock Center, Yale University, and is described in Datsenko etal., Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)). The amplified desAgene (described above) was digested with AvrII and EcoRI. TheAvrII-EcoRI fragment of the desA gene and the EcoRI-BamHI fragment ofthe kan gene were then inserted between the AvrII-BamHI sites ofpCDFDuet-1 (from EMD Chemicals, Gibbstown, N.J.) to produce a plasmidthat was named pCDFDuet-1-Δ5-kan.

A p84.17fabBΔ5kan plasmid was constructed to replace fabB with thedesA_kan cassette by several subcloning steps. First, a DNA fragment(L-fabB) flanking the upstream region of fabB was amplified with primersfabBLF and fabBLR (see Table 11), and a DNA fragment (R-fabB) flankingthe downstream region of fabB was amplified with primers fabBRF andfabBRR (see Table 11) from E. coli MG1655 genomic DNA. Second, L-fabBwas digested with XbaI and BglII, and R-fabB was digested with Nod andBglII. The digested L-fabB and R-fabB fragments were purified fromagarose gel and were ligated with XbaI-NotI linearized pKOV. Theresultant plasmid was designated pHZ1.186. Next, the desA_kan genecassette was removed from pCDFDuet-1-Δ5-kan as an AvrII-BamHI fragmentand was inserted between the AvrII and BglII sites of pHZ1.186,resulting in the desA_kan gene cassette being sandwiched by L-fabB andR-fabB. Finally, the L-fabB-desA_kan-R-fabB fragment was amplified withfabBLF and fabBRR (see Table 11) from pHZ1.186 and cloned into the twoPvuII sites of pMOD-4-MCS (Epicentre Biotechnologies, Madison, Wis.).The final plasmid was designated p84.17fabB.

DNA spanning from about 1 kb upstream to about 1 kb downstream offabB::cm was amplified from the genome of GRT23 cells using the primersfabBup and fabBdowm (see Table 11). The amplified DNA fragment was thendigested with PvuII and inserted between the two PvuII sites ofpMOD-4-MCS. The resulting plasmid was designated p84.15.

The genes encoding a thioesterase ('TesA) and a fatty acid reductase(CarB) were cloned as an operon, and the operon was placed under the trcpromoter and pCL 1920 vector. The final plasmid was namedpCL-Ptrc-carB_'tesA (the sequence is listed in FIG. 17 as SEQ IDNO:213).

TABLE 11 Primer sequences Primer ID Sequence delta5Fn TTTT CCTAGG ATGACT GAA CAA ACC A (SEQ ID NO: 214) delta5Rn TTTT GAATTC TTA TCA TTG TGAAAG CCAGAA (SEQ ID NO: 215) kanF TTTT GAATTC TGT AGG CTG GAG CTG CTTCG(SEQ ID NO: 216) kanR ATTCCG GGG ATC CGT CGA CC (SEQ ID NO: 217) fabBLFTTTT CTA GAA ATA GCG CCA GCG ACA (SEQ ID NO: 218) fabBLR TTTT AGA TCTTAG CCC TAG GCC AGT AAT CAC TGC ACG (SEQ ID NO: 219) fabBRF TTTT AGA TCTAGC TTC GGC TTC GGC G (SEQ ID NO: 220) fabBRR TTTT GCG GCC GCG CCC ATCCTT TGC TGG C (SEQ ID NO: 221) fabBup ACG ACA AAT GCG CCG C (SEQ ID NO:222) fabBdown ATC CGC GCA ATA AAG C (SEQ ID NO: 223)Strain Construction

An E. coli MG1655 (ΔfadEΔfhuAfabB::cm)/pACYC-Δ5 strain was constructedby transforming p84.15fabB into MG1655 (ΔfadEΔfhuA)/pACYC-Δ5. Plasmidp84.17fabB was transformed into MG1655 (ΔfadEΔfhuAfabB::cm)/pACYC-Δ5 toproduce MG1655 (ΔfadEΔfhuAfabB::desA_kan)/pACYC-Δ5. After eachtransformation, the transformant mix was plated onto L agar platessupplemented with 1 mM IPTG and appropriate antibiotics (17 mg/L ofchloramphenicol or 50 mg/L of kanamycin).

MG1655 (ΔfadEΔfhuAfabB::desA_kan)/pACYC-Δ5 grew normally in L Brothsupplemented with oleate (potassium salt, 50 mg/L). Cells were platedonto L agar plates supplemented with 50 mg/L of oleate and incubated at37° C. for 2 days. Colonies were then patched onto L Agar plates,supplemented with 50 mg/L of oleate and 100 mg/L of carbenicillin. Oneof the colonies, which lost resistance to carbenicillin but retainedkanamycin resistance, was streaked onto an L agar plate supplementedwith 50 mg/L of kanamycin, but no oleate. One of the colonies wasselected from the plate and was designated ALC119A.

ALC119A with a Fatty Alcohol Pathway Produced Almost Exclusive SaturatedFatty Alcohol

Plasmid pCL-Ptrc-carB_'tesA was transformed into the ACL119A strain.Three transformants of ALC119A/pCL-Ptrc-carB_'tesA were grown in 3 mL ofL broth with 100 mg/L of spectinomycin in a 37° C. shaker overnight. 15μL of the overnight culture were transferred into 2 mL of fresh L brothwith 100 mg/L of spectinomycin and 2 μL of 70% potassium oleate. Thefresh inoculation was placed in a 37° C. shaker for about 3 hrs. The 2mL culture was then transferred into 20 mL of V9 medium (Hu-9 mediumwithout ferric chloride) in a 125 mL baffle flask. When the OD₆₀₀ of theculture reached about 0.9, 1 mM of IPTG was added to each flask. After20 hrs of growth at 37° C., 20 mL of ethyl acetate (with 1% of aceticacid, v/v) was added to each flask to extract the fatty alcoholsproduced during the fermentation. The crude ethyl acetate extract wasdirectly analyzed with GC/MS as described in WO 2008/119082. Cetylalcohol was used as a reference for quantification of fatty alcohol.

As shown in FIG. 13, the ALC119A/pCL-Ptrc-carB_'tesA strain producedalmost exclusively saturated fatty alcohols, including dodecanol,tetradecanol and hexadecanol.

Example 6 Production of Fatty Alcohols in the CyanobacteriumSynechococcus sp. PCC7002

This example describes the use of photoautotrophic bacteria to producefatty alcohols from carbon dioxide (instead of glucose) using thecarB-'tesA-yahK pathway. First, a vector is constructed for homologousrecombination into the Synechococcus sp. PCC7002 plasmid pAQ1 (genbankaccession NC_(—)0050525) using 500 by homologous regions correspondingto positions 3301-3800 and 3801-4300 of pAQ1. As a selectable marker, aspectinomycin resistance cassette containing the aminoglycoside 3′adenylyltransferase, aad, promoter, gene and terminator (from plasmidpCL1920), is added between the homologous regions. For gene expression,the promoter and ribosome binding site of aminoglycosidephosphotransferase, aph (from plasmid pACYC177), is added followed bythe unique cloning sites NdeI and EcoRI for insertion of a heterologousgene or operon. This complete integration cassette is constructed bygene synthesis and cloned into pUC19 for maintenance and delivery. Theresulting plasmid, pLS9-7002, allows (i) cloning and expression of aforeign gene, and (ii) delivery and stable in vivo integration intoSynechococcus sp. PCC7002 plasmid pAQ1.

The fatty alcohol pathway for expression in Synechococcus sp. PCC7002 isconstructed as follows. The carB-'tesA operon from pCL-Ptrc-carB-'tesA(described in Example 4) is extended by adding yahK downstream of 'tesAand then cloning into the NdeI and EcoRI sites of pLS9-7002 downstreamof the aph promoter and ribosome binding site. The resulting plasmid istransformed into Synechococcus sp. PCC7002 as described by Stevens etal. (Proc. Natl. Acad. Sci. U.S.A. 77:6052-6056 (1980)). Stableintegrants are selected for on ATCC 1047 medium supplemented with 15μg/mL spectinomycin. 1 L of ATCC 1047 medium contains 40 mg MgSO₄×7H₂O,20 mg CaCl₂×2H₂O, 750 mg NaNO₃, 2 mg K₂HPO₄, 3.0 mg citric acid, 3.0 mgferric ammonium citrate, 0.5 mg EDTA, 20 mg Na₂CO₃, 2.86 mg H₃BO₃, 1.81mg MnCl₂, 0.22 mg ZnSO₄, 0.04 mg Na₂MoO₄, 0.08 mg CuSO₄, 0.05 mgCo(NO₃)₂, 0.02 mg vitamin B12, 10 g agar, and 750 mL sea water.Spectinomycin resistant colonies are restreaked several times on ATCCmedium 1047 with spectinomycin and tested for isogenic intergration ofthe carB-'tesA-yahK operon by PCR with primers pAQ1-U(atgtctgacaaggggtttgacccct) (SEQ ID NO:224) and pAQ1-D(gcacatccttatccaattgctctag) (SEQ ID NO:225). Complete isogeniccarB-'tesA-yahK integrants are then grown in 50 mL liquid ATCC 1047medium with spectinomycin in 500 mL shake flasks with appropriateaeration and illumination at 30° C. for five to seven days. Culturealiquots are extracted at various time points with an equal volume ofethyl acetate and the extracts are analyzed for fatty alcohol productionas described in Example 3. Fatty alcohols are produced.

Example 7 Production of Fatty Alcohols in the CyanobacteriumSynechococcus elongatus PCC7942

This example describes a second method of using photoautotrophicbacteria to produce fatty alcohols from carbon dioxide (instead ofglucose) using the carB-'tesA-yahK pathway. First, a vector isconstructed for homologous recombination into the Synechococcuselongatus PCC7942 genome (genbank accession CP_(—)000100) using 800 byhomologous regions corresponding to positions 2577844-2578659 and2578660-2579467 of CP_(—)000100. This chromosomal location is known asneutral site one (NS1) (Mackey et al., Meth. Mol. Biol. 362:115-129(2007)). As a selectable marker, a spectinomycin resistance cassettecontaining the aminoglycoside 3′ adenylyltransferase, aad, promoter,gene and terminator (from plasmid pCL1920), is added between thehomologous regions. Additionally, the unique cloning sites NdeI andEcoRI are added for insertion of a heterologous gene or operon. Thisintegration cassette is constructed by gene synthesis and cloned intopUC19 for maintenance and delivery. The resulting plasmid,pLS9-7942_NS1, allows (i) cloning and expression of a foreign gene and(ii) delivery and stable in vivo integration into the Synechococcuselongatus PCC7942 genome.

The complete carB-'tesA-yahK operon (described in Example 6), includingits ptrc promoter and ribosome binding site, is cloned into the NdeI orEcoRI site of pLS9-7942_NS1. The resulting plasmid is transformed intoS. elongatus PCC7942 as described by Mackey et al., Meth. Mol. Biol.362:115-129 (2007). Stable integrants are selected for on BG-11 mediumsupplemented with 4 μg/mL spectinomycin. 1 L of BG-11 medium contains 75mg MgSO₄×7H₂O, 36 mg CaCl₂×2H₂O, 1.5 g NaNO₃, 40 mg K₂HPO₄, 6.0 mgcitric acid, 6.0 mg ferric ammonium citrate, 1.0 mg EDTA, 20 mg Na₂CO₃,2.86 mg H₃BO₃, 1.81 mg MnCl₂, 0.22 mg ZnSO₄, 0.04 mg Na₂MoO₄, 0.08 mgCuSO₄, 0.05 mg Co(NO₃)₂, and 10 g agar. Spectinomycin resistant coloniesare restreaked several times on BG-11 medium with spectinomycin andtested for isogenic integration of the carB-'tesA-yahK operon by PCRwith primers NS1-U (gatcaaacaggtgcagcagcaactt) (SEQ ID NO:226) and NS1-D(attcttgacaagcgatcgcggtcac) (SEQ ID NO:227). Complete isogeniccarB-'tesA-yahK integrants are then grown in 50 mL liquid BG-11 mediumwith spectinomycin in 500 mL shake flasks with appropriate aeration andillumination at 30° C. up to seven days. Culture aliquots are extractedat various time points with an equal volume of ethyl acetate and theextracts are analyzed for fatty alcohol production as described inExample 3. Fatty alcohols are produced.

Example 8 Malonyl-CoA-Independent Production of Fatty Alcohols in E.coli

Certain protists such as Euglena gracilis are capable of malonyl-CoAindependent fatty acid biosynthesis. The biosynthetic machinery for thispathway is located in the mitochondria and is thought to reverse thedirection of β-oxidation by using acetyl-CoA as priming as well aselongating substrates to produce C₈ to C₁₈ fatty acids (Inui et al.,Eur. J. Biochem. 142:121-126 (1984)). The enzymes involved aretrans-2-enoyl-CoA reductases (TER), which catalyze the irreversiblereduction of trans-2-enoyl-CoA to acyl-CoA and thereby drive theotherwise reversible pathway in the reductive direction (while theopposite is true for β-oxidation, where the irreversible acyl-CoAdehydrogenase, FadE, drives the reaction in the oxidative direction).One TER gene from E. gracilis as well as other eukaryotic andprokaryotic homologs are known (Hoffmeister et al., J. Biol. Chem.280:4329-4338 (2005); Tucci et al., FEBS Lett. 581:1561-1566 (2007)).The only known TER enzyme from E. gracilis has been shown in vitro toreduce trans-2-butenoyl-CoA (C4) and trans-2-hexenoyl-CoA (C6) to therespective acyl-CoAs, (longer-chain trans-2-enoyl-CoAs have not beentested). Currently, very little is known about the other pathway enzymesin E. gracilis.

A pathway that creates a flux exclusively from acetyl-CoA precursors toacyl-CoA (as in Euglena gracilis mitochondria) can be engineered in E.coli using different sets of enzymes with the following four enzymaticactivities: (i) non-decarboxylating, condensing thiolase, (ii)3-ketoacyl-CoA reductase (or 3-hydroxyacyl-CoA dehydrogenase), (iii)3-hydroxyacyl-CoA hydratase (or enoyl-CoA dehydratase) and (iv)trans-2-enoyl-CoA reductase. All four enzymes can have sufficientlyrelaxed chain lengths specificity to allow synthesis of acyl-CoAs withlonger chain length, e.g., C₁₂ or C₁₄.

A plasmid encoding all four activities is constructed as follows. Asynthetic operon of E. coli fadA (YP_(—)026272) (shown in FIG. 17 as SEQID NO:229) (non-decarboxylating thiolase) and fadB (NP_(—)418288) (shownin FIG. 17 as SEQ ID NO:231) (3-hydroxyacyl-CoA dehydrogenase andenoyl-CoA dehydratase) and E. gracilis ter (Q5EU90) (shown in FIG. 17 asSEQ ID NO:228) (trans-2-enoyl-CoA reductase, codon optimized without its5′ sequence encoding a transit peptide) is constructed and cloneddownstream of a ptrc promoter into a pACYC plasmid with a carbenicillinor chloramphenicol resistance gene. Alternatively, instead of the E.coli fadA and fadB genes, the E. colifadI (NP_(—)416844) (shown in FIG.17 as SEQ ID NO:223) and fadJ (NP_(—)416843) (shown in FIG. 17 as SEQ IDNO:235) genes (or the corresponding orthologs from other organisms) areused. As an alternative to the E. gracilis ter gene, the correspondingorthologs from other organisms or the E. coli fabI (NP_(—)415804) (shownin FIG. 17 as SEQ ID NO:237) gene are used. Although FabI normallyreduces trans-2-enoyl-ACPs, it is also active with trans-2-enoyl-CoAs(Bergler et al., J. Biol. Chem. 269:5493-5496 (1993)).

The pACYC-ptrc_fadAB-ter plasmid or the pACYC-ptrc_fadAB-fabI plasmid iscotransformed with the pCL-ptrc_carB-'tesA plasmid (described in Example4) into an E. coli ΔfadE strain. These strains are cultured, extractedand analyzed for fatty alcohol production as described in Example 3. Thetwo different strains produce fatty alcohols with different chain lengthdistribution.

As these strains express 'TesA, a portion of the fatty alcohols producedare derived from malonyl-CoA dependent acyl-ACP precursors. 'TesAefficiently hydrolyzes acyl-ACPs when overexpressed in E. coli, althoughit has higher specific activity for acyl-CoAs as compared to acyl-ACPs.To increase the proportion or exclusively produce fatty alcohols derivedfrom the malonyl-CoA independent pathway, alternative thioesterases thathave lower hydrolytic activity towards acyl-ACPs are used instead of'TesA. One example is E. coli TesB (NP_(—)414986), which prefersacyl-CoAs over acyl-ACPs (Spencer et al., J. Biol. Chem. 253:5922-5926(1978)) and when overexpressed in E. coli does not hydrolyze acyl ACPs(Zheng et al., App. Environ. Microbiol. 70:3807-3813 (2004)). Inalternative methods, orthologs of TesA and TesB or thioesterases fromother protein families that hydrolyze acyl-CoAs with high efficiencywhile hydrolyzing acyl-ACPs with low efficiency are used.

In one method, a pCL-ptrc_carB-'tesB plasmid is constructed as describedin Example 4 by replacing the 'tesA gene with the tesB gene(NP_(—)414986) (shown in FIG. 17 as SEQ ID NO:239). The plasmid iscotransformed with the pACYC-ptrc_fadAB-ter plasmid or thepACYC-ptrc_fadAB-fabI plasmid into an E. coli ΔfadE strain. Thesestrains are cultured, extracted and analyzed for fatty alcoholproduction as described in Example 3.

In another method, the pCL-ptrc_carB-'tesA plasmid is replaced with apCL-ptrc_acr1 plasmid, which expresses the acyl-CoA reductase Acr1 fromAcinetobacter baylyi ADP1 (YP_(—)047869) (shown in FIG. 17 as SEQ IDNO:241). This reductase specifically reduces acyl-CoAs but not acyl-ACPsto the corresponding fatty alcohols (Reiser et al., J. Bacteriol.179:2969-2975 (1997)). The plasmid is cotransformed with thepACYC-ptrc_fadAB-ter plasmid or the pACYC-ptrc_fadAB-fabI plasmid intoan E. coli ΔfadE strain. These strains are cultured, extracted andanalyzed for fatty alcohol production as described in Example 3. Thestrains produce fatty alcohols independent of malonyl-CoA.

Example 9 Identification of Iron as an Inhibitor of Fatty AlcoholProduction

Hu9 medium is a known fermentation medium, which contains 6 g/L Na₂HPO₄,3 g/L KH₂PO₄, 0.5 g/L NaCl, 1 g/L NH₄Cl, 0.1 mM CaCl₂, 1 mM MgSO₄, 15g/L agar, 10 mM glucose, 50 mg/L Uracil, and trace minerals containing100 μM FeCl₃, 500 μM ZnCl₂, 200 μM Na₂Mo₄, 200 μM CuSO₄, 200 μM H₃BO₃.However, it was observed that the production of fatty alcohols wascompletely reduced when recombinant E. coli strains, otherwise capableof producing fatty alcohols, were grown in Hu9 medium. As described indetail below, the inability of E. coli strains to produce fatty alcoholsin various incomplete Hu9 media was measured, and it was found that therecombinant bacteria were incapable of producing fatty alcohols wheniron was present in the medium. However, the addition of iron did notinhibit the growth of the bacteria.

In order to identify the component(s) involved in the inhibition offatty alcohol production, different versions of incomplete Hu9 mediumwere made, some of which lacked a dispensable ingredient, and then theproduction of fatty alcohol was evaluated.

In the first step the following media were made: complete Hu9 medium,incomplete Hu9 medium lacking uracil, and incomplete Hu9 medium lackingtrace elements. K6 cells (a recombinant bacterial strain C41 (DE3,ΔfadE) carrying pACYCDuet-1-carB, encoding the CAR homolog carB andpETDuet-1-'tesA) were cultured in 2 mL of LB containing appropriateantibiotics. After reaching an OD of 1.0, the 2 ml, cultures were scaledup in 125 mL shake flasks (containing one of the Hu9 media describedabove) to a volume 22 mL. The cultures were induced by adding IPTG to afinal concentration of 1 mM. After growing them for 20 hrs at 37° C., 22mL of ethyl acetate (with 1% of acetic acid, v/v) was added to eachflask to extract the fatty alcohols produced during the fermentation.The crude ethyl acetate extract was directly analyzed with GC/MS and thetotal fatty alcohol titers were quantified.

As depicted in FIG. 14A, the fatty alcohol production was inhibited to agreat extent by the addition of trace elements as compared to theaddition of uracil to the incomplete Hu9 medium. This indicated that theinhibitory component(s) was a part of trace mineral solution.

In order to find out which trace element was responsible for the fattyalcohol production inhibition, the following Hu9 media were made:complete Hu9 medium; Hu9 lacking FeCl₃; Hu9 lacking ZnCl₂; Hu9 lackingNa₂Mo₄; Hu9 lacking CuSO₄; and Hu9 lacking H₃BO₃. The fatty alcoholproduction of K6 cells grown in these different Hu9 media was evaluatedusing the method described above.

As shown in FIG. 14B, fatty alcohol production was inhibited mainly bythe addition of iron to the medium. Thus, by eliminating or reducing thepresence of iron (e.g., ferric citrate, ferric chloride, or ferroussulfate) in the culture medium, fatty alcohols can be produced.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

The invention claimed is:
 1. A method of producing a fatty alcoholcomposition comprising C12 and C14 fatty alcohols in a bacterial cell,the method comprising: (a) expressing in the bacterial cell a geneencoding a polypeptide having thioesterase (EC 3.1.2- or EC 3.1.1.5)activity effective to release a fatty acid from acyl-ACP; and (b)further expressing in said bacterial cell an exogenous gene encoding apolypeptide having the amino acid sequence of SEQ ID NO:22; and (c)culturing said bacterial cell in culture media containing a carbohydratecarbon source under conditions wherein at least 25 mg of the compositionis produced per liter of bacterial culture.
 2. The method of claim 1,further comprising expressing a gene encoding a polypeptide havingalcohol dehydrogenase (EC 1.1.1.*) activity in the bacterial cell,wherein said polypeptide converts a fatty aldehyde to a fatty alcohol.3. The method of claim 1, wherein the bacterial cell expresses anattenuated level of at least one fatty acid degradation enzyme relativeto a wild type bacterial cell.
 4. The method of claim 3, wherein the atleast one fatty acid degradation enzyme has acyl-CoA synthetaseactivity.
 5. The method of claim 1, wherein the composition comprises aC12 fatty alcohol, C14:1 fatty alcohol, C14:0 fatty alcohol, C16:0 fattyalcohol, and C16:1 fatty alcohol.
 6. The method of claim 5, wherein theC12 fatty alcohol, C14:1 fatty alcohol, C14:0 fatty alcohol, C16:0 fattyalcohol, and C16:1 fatty alcohol are each a primary alcohol.
 7. Themethod of claim 1, wherein the composition has a C12 to C14 ratio ofapproximately 3 to
 1. 8. The method of claim 1, wherein the compositioncomprises an unsaturated fatty alcohol.
 9. The method of claim 8,wherein the unsaturated fatty alcohol is C10:1, C12:1, C14:1, C16:1, orC18:1.
 10. The method of claim 9, wherein the fatty alcohol isunsaturated at the omega-7 position.
 11. The method of claim 8, whereinthe unsaturated fatty alcohol comprises a cis double bond.
 12. Themethod of claim 1, wherein the composition comprises a saturated fattyalcohol.
 13. The method of claim 1, further comprising culturing thebacterial cell in the presence of at least one biological substrate forthe polypeptide set forth in SEQ ID NO:22.
 14. The method of claim 13,wherein the at least one substrate is a C12 or C14 fatty acid.
 15. Themethod of claim 1, wherein said fatty alcohol is produced by way of amalonyl-CoA-independent pathway.
 16. The method of claim 1, wherein thecarbohydrate carbon source comprises a monosaccharide, disaccharide orpolysaccharide.
 17. The method of claim 1, further comprising isolatingthe fatty alcohol composition from the bacterial cell culture.
 18. Amethod of producing a fatty alcohol composition comprising C12 and C14fatty alcohols in a bacterial cell, the method comprising: culturing thebacterial cell at 37° C. in a culture media containing a carbohydratecarbon source, wherein the bacterial cell comprises polynucleotidesequences encoding: (a) a first polypeptide having thioesterase (EC3.1.2.14 or EC 3.1.1.5) activity effective to release a fatty acid fromacyl-ACP, and (b) a second polypeptide having carboxylic acid reductaseactivity and comprising an amino acid sequence (i) having at least 95%sequence identity to SEQ ID NO:22, and (ii) having activity effective toconvert a fatty acid to a fatty aldehyde, wherein the second polypeptideis expressed from an exogenous gene, wherein the second polypeptidecomprises SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9 and SEQ ID NO: 10; andwherein the composition is produced at a yield of at least 300 mg/L ofbacterial culture.
 19. The method of claim 18, wherein one or more ofsaid polynucleotide sequences are plasmid sequences.
 20. The method ofclaim 18, wherein one or more of the polynucleotide sequences areintegrated into the genome of the bacterial cell.
 21. The method ofclaim 18, wherein the second polypeptide comprises the amino acidsequence of SEQ ID NO:
 22. 22. The method of claim 18, wherein the firstpolypeptide is a TesA polypeptide.
 23. The method of claim 18, whereinthe composition comprises C12 and C14 fatty alcohols that are primaryalcohols.
 24. The method of claim 18, wherein the composition comprisesan unsaturated fatty alcohol.
 25. The method of claim 24, wherein thefatty alcohol is unsaturated at the omega-7 position.
 26. The method ofclaim 24, wherein the unsaturated fatty alcohol comprises a cis doublebond.
 27. The method of claim 18, wherein the composition furthercomprises a C6, C8, C10, C13, C15, C16, C11, or C18 fatty alcohol. 28.The method of claim 18, wherein the fatty alcohol has a C12 to C14 ratioof approximately 3 to
 1. 29. The method of claim 18, wherein thebacterial cell further comprises a polynucleotide sequence encoding athird polypeptide, the third polypeptide having alcohol dehydrogenase(EC 1.1.1.*) activity effective to convert a fatty aldehyde to a fattyalcohol.
 30. The method of claim 29, wherein the composition is producedat a level of at least 700 mg/L.
 31. The method of claim 18, wherein thecarbohydrate carbon source comprises a monosaccharide, disaccharide orpolysaccharide.